Cortistatin: neuropeptides

ABSTRACT

The present invention relates generally to nucleic acids encoding a novel neuropeptide designated cortistatin. The cortistatin nucleic acids, proteins and polypeptides thereof along with anti-cortistatin antibodies are useful in both screening methods, diagnostic methods and therapeutic methods related to modulation of sleep and disorders thereof.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of U.S. Ser. No. 08/648,322,filed May 15, 1996, now U.S. Pat. No. 6,074,872, pending, the disclosureof which is incorporated by reference herein.

GOVERNMENT SUPPORT

This invention was made with the support of the United States Governmentand the United States Government has certain rights in the inventionpursuant to the United States Public Health Service Contract GM32355 andNS22111.

TECHNICAL FIELD

The present invention relates generally to the discovery of aneuropeptide, designated cortistatin, that shares structural similaritywith somatostatin yet, unlike somatostatin, enhances slow-wave sleep.Cortistatin nucleic acid and encoded polypeptides along withanti-cortistatin antibodies are useful in both screening methods,diagnostic methods and therapeutic methods related to modulation ofsleep and disorders thereof.

BACKGROUND

Changes in arousal state from waking. to sleep are accompanied bydramatic changes in the electroencephalogram (EEG). The low amplitude,high frequency pattern of the awake EEG becomes dominated by highamplitude, low frequency synchronized activity in slow-wave sleep (SWS),followed sequentially by rapid eye movement (REM) sleep (Steriade etal., Science, 262:679-685 (1993). Acetylcholine (ACh) plays a key rolein the transition of the different phases of sleep (Shiromani et al.,Ann. Rev. Pharmacol. Toxicol., 27:137-156 (1987). SWS requires low AChlevels whereas REM sleep is characterized by high ACh content. Also,these phases of sleep have been shown to be differentially sensitive toa number of endogenous neuropeptides and cytokines, includingsomatostatin, which is known to increase REM sleep without significantlyaffecting other phases (Borbely et al., Physiol. Rev., 69:605-670(1989).

The present invention describes the cloning and characterization ofcortistatin, a novel neuropeptide that has been discovered to be asleep-modulating molecule with effects opposing those mediated bysomatostatin. Cortistatin, however, exhibits strong structuralsimilarity to somatostatin. Thus obtaining a cDNA clone from screeningbrain-specific libraries, the mRNA of which clone is translated into anaturally occurring physiologically active protein, is yet a furtherexample of such molecules described in U.S. Pat. Nos. 4,900,811 and5,242,798.

Although cortistatin has now been determined to be the product of adifferent gene, because of its structural similarity to somatostatin aswell as functional aspects described herein, cortistatin is a new memberof the somatostatin family whose distribution is primarily restricted toGABAergic cortical interneurons.

GABAergic neurons have been shown to finely modulate the output ofprincipal neurons of the cerebral cortex and hippocampus (Buhl et al.,Nature, 368:823-828 (1994), areas that have been implicated in arousalstate and complex cognitive functions, including learning and memory(Wilson et al., Science, 265:676-679 (1994).

The neuropeptide somatostatin was first described as a hypothalamicpeptide that inhibited growth hormone release (Brazeau et al., Science,179:77-79 (1973), and has since been implicated in many physiologicalphenomena, including hippocampal function and REM sleep generation(Danguir, Brain Res., 367:26-30 (1986). In the hippocampus, somatostatinis present largely in a particular set of interneurons. See, Hendry etal., Proc. Natl. Acad. Sci., USA, 81:6526-6530 (1984); Schemchel et al.,Neurosci. Lett., 47:227-232 (1984); and Morrison et al., Brain Res.,262:344-351 (1983). Somatostatin may modulate the output of pyramidalneurons primarily by depressing neuronal excitability, in part viaenhancement of the voltage-dependent potassium M current. See, Moore etal., Science, 239:278-280 (1988) and Schweitzer et al., Nature,346:464-466 (1990). Pharmacological studies have shown that somatostatinalso interacts with cholinergic (Araujo et al., J. Neurochem.,55:1546-1555 (1990) and Mancillas et al., Proc. Natl. Acad. Sci., USA,83:7518-7521 (1986) and GABAergic (Freund et al., Nature, 336:170-173(1988) systems, among others, thus modulating systems thought tounderlie different aspects of behavior.

As shown in the present invention, despite the physical similaritiesbetween somatostatin and cortistatin, administration of cortistatin invivo depresses neuronal electrical activity but, unlike somatostatin,induces low frequency waves in the cerebral cortex and antagonizes theeffects of acetylcholine on hippocampal and cortical measures ofexcitability, thus providing a mechanism for cortical synchronizationrelated to sleep.

BRIEF SUMMARY OF THE INVENTION

A mammalian neuropeptide, designated cortistatin, has now beendiscovered, cloned, sequenced and characterized for biological activity.Cortistatin is expressed in cortical and hippocampal mammalianinterneurons, has an amino acid residue sequence similar to but distinctfrom somatostatin, and has neurologic properties including neuronaldepression, sleep modulation and enhanced slow wave sleep.

The basic discovery of a new polypeptide of this nature provides avariety of embodiments, including compositions, methods of their use,and screening procedures for the identification of additional usefulcompositions.

In one embodiment, the invention describes a substantially isolatedcortistatin protein and a cortistatin polypeptide including an aminoacid residue sequence defining a cortistatin polypeptide having asequence that corresponds to a sequence in the Sequence Listing selectedfrom the group consisting of SEQ ID NOs 2, 5, 6, 7, 8, 9, 10, 11, 12,23, 24, 26, positions 44 to 74 of SEQ ID NO 26, positions 77 to 105 ofSEQ ID NO 26, and positions 89 to 105 of SEQ ID NO 26. The polypeptidecan be synthetic, recombinant or a fusion protein. Polypeptide analogsof cortistatin are also described.

The invention also describes a substantially purified nucleic acidhaving a nucleotide sequence that encodes a cortistatin polypeptidehaving a sequence that corresponds to a sequence in the Sequence Listingselected from the group consisting of SEQ ID NOs 2, 5, 6, 7, 8, 9, 10,11, 12, 23, 24, 26, positions 44 to 74 of SEQ ID NO 26, positions 77 to105 of SEQ ID NO 26, and positions 89 to 105 of SEQ ID NO 26. Thenucleic acid can be operatively linked to a promoter in an expressionvector. Vectors for expressing cortistatin and cells containing thevectors are also described. Polynucleotide primers useful forhybridizing to cortistatin genes and gene products (e.g., mRNA) are alsodescribed.

The invention also contemplates an antibody that immunoreacts withcortistatin or with a polypeptide having a sequence that corresponds toa sequence in the Sequence Listing selected from the group consisting ofSEQ ID NOs 2, 5, 6, 7, 8, 9, 10, 11, 12, 23, 24, 26, positions 44 to 74of SEQ ID NO 26, positions 77 to 105 of SEQ ID NO 26, and positions 89to 105 of SEQ ID NO 26. The antibody can also be a monoclonal antibody.

The invention also contemplates a kit for detecting the presence ofcortistatin in a human body sample comprising an anti-cortistatinantibody, cortistatin polypeptide or oligonucleotide of the invention.

The invention further contemplates methods for detecting the presence ofa nucleic acid that encodes cortistatin in a human body samplecontaining nucleic acid comprising the steps of:

(a) hybridizing the nucleic acid in the body sample with aoligonucleotide that includes at least 10 contiguous nucleotides fromthe nucleotide sequence shown in SEQ ID NO 1 from nucleotide 324 tonucleotide 366 to form a hybridization product; and

(b) detecting the presence of the hybridization product.

In a related method the invention describes a method of detecting thepresence of a cortistatin antigen in a human body sample comprising thesteps of:

(a) contacting a human body sample with an anti-cortistatin antibodythat immunoreacts with human cortistatin or with a polypeptide havingthe amino acid residue sequence shown in SEQ ID NO 8 for a time periodsufficient for said antibody to immunoreact with said antigen present inthe sample and form an immunoreaction complex; and

(b) detecting the presence of an immunoreaction complex, therebydetecting said antigen.

Screening methods for identifying a ligand that binds to cortistatinreceptor are also described which comprise:

(a) contacting a mammalian cell having a cortistatin receptor with acandidate ligand under conditions permitting binding of a knowncortistatin receptor ligand to said cortistatin receptor; and

(b) detecting the presence of any of said candidate ligand bound to saidreceptor, or:

(a) contacting a mammalian cell having a cortistatin receptor with acandidate ligand under conditions permitting binding of a knowncortistatin receptor ligand to said cortistatin receptor in the presenceof a labeled cortistatin receptor ligand; and

(b) detecting the presence of any of said labeled ligand bound to saidreceptor.

Cortistatin polypeptides can also be used to directly detect thepresence of a cortistatin receptor in a tissue sample comprising thesteps of:

(a) contacting a tissue sample with an isolated cortistatin ligand underconditions permitting binding of a known cortistatin ligand to saidcortistatin receptor; and

(b) detecting the presence of isolated cortistatin ligand bound to saidtissue sample.

Therapeutic methods for altering cortistatin gene expression in a cellare contemplated comprising introducing into said cell anoligonucleotide capable of specifically hybridizing to the cortistatingene. Alternatively, a method for activating the physiological responseof cortistatin receptor upon binding to cortistatin is contemplatedcomprising contacting said cortistatin receptor with a pharmaceuticalcomposition comprising a physiologically acceptable carrier and aneffective activating amount of a cortistatin receptor agonist.Similarly, a cortistatin receptor antagonist can be used to inhibit thereceptor.

Mutations in the cortistatin gene of a mammal that comprises anexpansion of the CTG domain of the cortistatin gene can be assayed,comprising the steps of:

(a) determining the nucleotide sequence of the CTG domain of thecortistatin gene in a nucleic acid sample from said mammal; and

(b) comparing the determined nucleotide sequence to the known sequenceof the CTG domain in a normal cortistatin gene to identify the presenceof a sequence expansion in the CTG domain, and thereby said mutation.

The pharmacological activity of a cortistatin polypeptide can beexploited in a method for inducing sleep in a mammal comprisingadministering a physiologically tolerable composition containing atherapeutically effective amount of a cortistatin analog to said mammal.Similarly, sleep can be inhibited by use of a cortistatin receptorantagonist.

Other embodiments will be apparent to one skilled in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the nucleotide sequence and predicted amino acidsequence of rat cortistatin as described in Example 1. Thepreprocortistatin cDNA clone displays a 336 nucleotide open readingframe with a N-terminal signal peptide whose cleavage site is indicatedby an arrow. The CTG repeat contained within the coding region for thesignal peptide is underlined. Two proteolytic cleavage sites (bold KK orKR) could give rise to peptides 13 (hatched lined box) and 14 aminoacids long (solid line box), or to the 29-residue precursor.

FIG. 2 illustrates the alignment of cortistatin-29 (CST) andsomatostatin-28 (SST) amino acid residue sequences as described inExample 1.

FIG. 3 illustrate the nucleotide sequence and predicted amino acidsequence of mouse cortistatin as described in Example 1. Thepreprocortistatin cDNA clone displays a 327 nucleotide open readingframe with a N-terminal signal peptide whose cleavage site is indicatedby an arrow. The CTG repeat contained within the coding region for thesignal peptide is underlined. Two proteolytic cleavage sites (KS or KK)could give rise to a 13 amino acid peptide (hatched lined box) and a 14amino acid peptide (solid line box).

FIG. 3a illustrates the alignment of the nucleotide sequences of rat,mouse and human preprocortistatin cDNAs. The human preprocortistatincDNA displays a 315 nucleotide open reading frame. The CTG repeat thatencodes the amino acid leucine, and that is of variable length betweenspecies has been underlined. Two possible polyadenylation signals aremarked with an asterisk. Nucleotides conserved among all three speciesare shown in uppercase, and those not conserved are shown in lowercase.

FIG. 3b illustrates the alignment of the deduced amino acid sequences ofthe rat, mouse and human cortistatin precusors. The putative dibasiccleavage sites are indicated in bold. Consensus residues are indicated.Proteolytic cleavage sites in human cortistatin (bold RR or RK) couldgive rise to a 31 amino acid peptide (hatched box), a 29 amino acidpeptide, and a 17 amino acid peptide(solid box).

FIG. 4 illustrates a photograph of a Northern blot containing twomicrograms of polyA+ selected RNA from rat brain, anterior pituitary,adrenal gland, liver, spleen, thymus, ovary and testes that washybridized with a cortistatin cDNA probe as described in Example 4.

FIG. 5A illustrates the displacement of ¹²⁵I-somatostatin bound to GH₄pituitary cells by the peptides, somatostatin-14 and cortistatin-14, asdescribed in Example 5. The displacement by cortistatin-14 is shown inthe filled circles while that by somatostatin-14 is shown in the whitecircles. the counts per minute per milligram of iodinated somatostatin(cpm/mg protein)×10⁻³ is listed on the Y-axis while the molarity (M) ofthe free peptides is listed on the X-axis. The combined data from fourindependent experiments are plotted as mean values ± standard error.Controls included TRH and VIP as described in Example 5.

FIG. 5B illustrates cyclic AMP stimulation in GH₄ cells followingtreatment with VIP, TRH, somatostatin-14 and cortistatin-14. The Y-axisplots the amount of intracellular cAMP levels in picomole per milligramof protein (pmol/mg protein)×10. The concentrations of the testedreagents are indicated on the X-axis. The results of the assay arediscussed in Example 5.

FIG. 5C illustrates inhibition of stimulated cAMP levels bysomatostatin-14 and cortistatin-14. The assay was performed as describedin Example 5. On the Y-axis, the change of cAMP concentration in pmol/mgprotein×10 is indicated against the concentration of the peptides foreither VIP or TRH induction on the X-axis. Each data point represents2-4 replicates and the experiments were carried out twice.

FIG. 6A illustrates the current-clamp recording of a CA1 neuron manuallydepolarized to −65 mV (resting membrane potential was −70 mV) to elicitaction potential firing (upward deflections, truncated) by superfusionwith 1 μM cortistatin-14 (bar above record). The assay and results arediscussed in Example 8.

FIG. 6B illustrates voltage-clamp recording of a CA1 neuron held at −43mV following treatment with cortistatin at 1 M for 7 minutes; an I_(M)relaxation was evoked with 10 mV hyperpolarizing step. Arrows indicatethe I_(M) relaxation amplitude while the dotted lines indicate anoutward steady-state control holding current. The assay and results arediscussed in Example 8.

FIG. 6C illustrates the effects of cortistatin-14 on population spike(PS) amplitudes in CA1 neurons in vivo. The assays and results arediscussed in Example 8. Stimulus response curves are indicated with thePS amplitude plotted on the Y-axis in mV against the stimulus levelplotted on the X-axis at three response levels: threshold, half-maximaland maximal (control mean half-maximal PS amplitude=4.7 mV±0.5; n=5).

FIGS. 7A-1 through 7A-4 illustrate the effect of theintracerebroventricular administration of cortistatin-14 on thesleep-wake cycle of the rat. FIGS. 7A-1, 7A-2, 7A-3 and 7A-4respectively show wakefulness, slow-wave sleep 1 (SWS1), slow-wave sleep2 (SWS2) and rapid eye movement (REM) sleep. The graphs all have themean+/−the standard error of the mean percent (%) of total time of therecording plotted on the Y-axis against the varying amount ofcortistatin-14 or control plotted on the X-axis. The assay and resultsare discussed in Example 8.

FIG. 7B illustrates the effects of iontophoretically appliedacetylcholine (ACh) (0.9 M), somatostatin-14 (1.5 mM) and cortistatin-14on PP responses in CA1 neurons in vivo. The percent of P2/P1 response isplotted on the Y-axis against the interstimulus interval in milliseconds(ms) plotted on the X-axis. The control is plotted with a empty circlewhile that with ACh is indicated with a dashed line and a filled square.The results with somatostatin-14 are indicated with a dashed line markedwith an open triangle and the combined ACh/cortistatin-14 treatment isindicated with a dashed line marked with a filled circle. The assay andresults are discussed in Example 8.

FIGS. 7C through 7F representative recordings of field potentialselicited in CA1 by commissural stimulation in 80 millisecond intervals.The baseline recording is plotted in FIG. 7C with the calibration barsof 2 mV and 10 ms. FIG. 7D shows the reduction of PP inhibition withiontophoretic administration of ACH. FIG. 7E shows that simultaneousapplication of cortistatin-14 with ACH antagonized the effect seen inFIG. 7D. FIG. 7F shows decreased PP inhibition obtained withsomatostatin-14 (calibration bars of 1 mV and 10 ms). The assay andresults are discussed in Example 8.

FIG. 7G illustrates the effects of microiontophoretically (100-250 nA)applied ACH and cortistatin-14 on local EEG activity recorded in thevisual cortex. The assay and results are discussed in Example 8. Theaveraged EEG power spectra is plotted on the Y-axis in mV2 against fastactivity of responses in baseline (filled column), ACH treatment(vertical lined-column), cortistatin-14 (dotted column) andACH/cortistatin-14 treated (empty column). The assay and results arediscussed in Example 8.

DETAILED DESCRIPTION OF THE INVENTION A. Definitions

Amino Acid Residue: An amino acid formed upon chemical digestion(hydrolysis) of a polypeptide at its peptide linkages. The amino acidresidues described herein are preferably in the “L” isomeric form.However, residues in the “D” isomeric form can be substituted for anyL-amino acid residue, as long as the desired functional property isretained by the polypeptide. NH₂ refers to the free amino group presentat the amino terminus of a polypeptide. COOH refers to the free carboxygroup present at the carboxy terminus of a polypeptide. In keeping withstandard polypeptide nomenclature (described in J. Biol. Chem.,243:3552-59 (1969) and adopted at 37 CFR §1.822(b) (2)), abbreviationsfor amino acid residues are shown in the following Table ofCorrespondence:

TABLE OF CORRESPONDENCE SYMBOL AMINO ACID 1-Letter 3-Letter Y Tyrtyrosine G Gly glycine F Phe phenylalanine M Met methionine A Alaalanine S Ser serine I Ile isoleucine L Leu leucine T Thr threonine VVal valine P Pro proline K Lys lysine H His histidine Q Gln glutamine EGlu glutamic acid Z Glx Glu and/or Gln W Trp tryptophan R Arg arginine DAsp aspartic acid N Asn asparagine B Asx Asn and/or Asp C Cys cysteine XXaa Unknown or other

It should be noted that all amino acid residue sequences representedherein by formulae have a left-to-right orientation in the conventionaldirection of amino terminus to carboxy terminus. In addition, the phrase“amino acid residue” is broadly defined to include the amino acidslisted in the Table of Correspondence and modified and unusual aminoacids, such as those listed in 37 CFR 1.822(b) (4), and incorporatedherein by reference. Furthermore, it should be noted that a dash at thebeginning or end of an amino acid residue sequence indicates a peptidebond to a further sequence of one or more amino acid residues or acovalent bond to an amino-terminal group such as NH₂ or acetyl or to acarboxy-terminal group such as COOH.

Recombinant DNA (rDNA) molecule: a DNA molecule produced by operativelylinking two DNA segments. Thus, a recombinant DNA molecule is a hybridDNA molecule comprising at least two nucleotide sequences not normallyfound together in nature. rDNA's not having a common biological origin,i.e., evolutionarily different, are said to be “heterologous”.

Vector: a rDNA molecule capable of autonomous replication in a cell andto which a DNA segment, e.g., gene or polynucleotide, can be operativelylinked so as to bring about replication of the attached segment. Vectorscapable of directing the expression of genes encoding for one or morepolypeptides are referred to herein as “expression vectors”.

Receptor: A receptor is a molecule, such as a protein, glycoprotein andthe like, that can specifically (non-randomly) bind to another molecule.

Antibody: The term antibody in its various grammatical forms is usedherein to refer to immunoglobulin molecules and immunologically activeportions of immunoglobulin molecules, i.e., molecules that contain anantibody combining site or paratope. Exemplary antibody molecules areintact immunoglobulin molecules, substantially intact immunoglobulinmolecules and portions of an immunoglobulin molecule, including thoseportions known in the art as. Fab, Fab′, F(ab′)₂ and F(v).

Antibody Combining Site: An antibody combining site is that structuralportion of an antibody molecule comprised of a heavy and light chainvariable and hypervariable regions that specifically binds (immunoreactswith) an antigen. The term immunoreact in its various forms meansspecific binding between an antigenic determinant-containing moleculeand a molecule containing an antibody combining site such as a wholeantibody molecule or a portion thereof.

Monoclonal Antibody: A monoclonal antibody in its various grammaticalforms refers to a population of antibody molecules that contain only onespecies of antibody combining site capable of immunoreacting with aparticular epitope. A monoclonal antibody thus typically displays asingle binding affinity for any epitope with which it immunoreacts. Amonoclonal antibody may therefore contain an antibody molecule having aplurality of antibody combining sites, each immunospecific for adifferent epitope, e.g., a bispecific monoclonal antibody. Althoughhistorically a monoclonal antibody was produced by immortalization of aclonally pure immunoglobulin secreting cell line, a monoclonally purepopulation of antibody molecules can also be prepared by the methods ofthe present invention.

Upstream: In the direction opposite to the direction of DNAtranscription, and therefore going from 5′ to 3′ on the non-codingstrand, or 3′ to 5′ on the mRNA.

Downstream: Further along a DNA sequence in the direction of sequencetranscription or read out, that is traveling in a 3′- to 5′-directionalong the non-coding strand of the DNA or 5′- to 3′-direction along theRNA transcript.

Reading Frame: Particular sequence of contiguous nucleotide triplets(codons) employed in translation that define the structural proteinencoding-portion of a gene, or structural gene. The reading framedepends on the location of the translation initiation codon.

Polypeptide: A linear series of amino acid residues connected to oneanother by peptide bonds between the alpha-amino group and carboxy groupof contiguous amino acid residues.

Protein: A linear series of greater than 50 amino acid residuesconnected one to the other as in a polypeptide.

Substantially Purified or Isolated: When used in the context ofpolypeptides or proteins, the terms describe those molecules that havebeen separated from components that naturally accompany them. Typically,a monomeric protein is substantially pure when at least about 60% to 75%of a sample exhibits a single polypeptide backbone. Minor variants orchemical modifications typically share the same polypeptide sequence. Asubstantially purified protein will typically comprise over about 85% to90% of a protein sample, more usually about 95%, and preferably will beover about 99% pure. Protein or polypeptide purity or homogeneity may beindicated by a number of means well known in the art, such aspolyacrylamide gel electrophoresis of a sample, followed byvisualization thereof by staining. For certain purposes, high resolutionis needed and high performance liquid chromatography (HPLC) or a similarmeans for purification utilized.

Synthetic Peptide: A chemically produced chain of amino acid residueslinked together by peptide bonds that is free of naturally occurringproteins and fragments thereof.

B. Cortistatin Proteins and Polypeptides

Cortistatin has been cloned, sequenced and characterized from a varietyof mammalian species, indicating that it is a neuropeptide found in allmammals, including humans, rodents, mice, and the like mammals. Theneuropeptide is not identical in amino acid residue sequence betweenmammalian species, but is sufficiently similar that allowsgeneralizations regarding function, and assures that one can identifyand isolate the cortistatin gene in any mammalian species.

Thus, variations at both the amino acid and nucleotide sequence levelare described in isolates of cortistatin, and such variations are not tobe construed as limiting. For example, allelic variation within amammalian species can tolerate a several percent difference betweenisolates of a type of cortistatin, which differences comprisenon-deleterious variant amino acid residues. Thus a protein of about 95%homology, and preferably at least 98% homology, to a disclosedcortistatin is considered to be an allelic variant of the disclosedcortistatin, and therefore is considered to be a cortistatin of thisinvention.

As disclosed herein, cortistatin is produced first in vivo in precursorform, and is then processed into smaller polypeptides having biologicalactivity as described herein. Insofar as these different polypeptideforms are contemplated as useful, the term cortistatin protein orpolypeptide connotes all species of polypeptide having an amino acidresidue sequence derived from the cortistatin gene.

The complete coding nucleotide sequence of rat preprocortistatin cDNA is438 nucleotides in length as shown in FIG. 1 and listed in SEQ ID NO 1.The complete preprocortistatin cDNA clone presents a 336 nucleotide openreading frame (ORF) with a N-terminal signal peptide whose cleavage sitebetween amino acid positions 27 and 28 corresponding to a cleavage siteafter nucleotide position 110.

Translation of this rat cDNA sequence encodes that a novel protein of112 amino acid residues, referred to as rat preprocortistatin. The aminoacid sequence of rat preprocortistatin is also listed in SEQ ID NO 1with the nucleotide sequence and in SEQ ID NO 2 alone.

Cleavage of the preprospecies to rat procortistatin produces a matureprotein that is processed at either of two tandem basic amino acidpairs, KK (lys-lys) or KR (lys-arg) to produce mature cortistatinproteins referred to as cortistatin-29 and cortistatin-14. This cleavagepattern is analogous to the cleavage of preprosomatostatin at 28 and 14residues as described by Glushankov et al., Proc. Natl. Acad. Sci., USA,81:6662-6666 (1984). Alternatively, cleavage at both basic pairs resultsin the production of mature cortistatin-13 in addition tocortistatin-14. The rat preprospecies along with prospecies and maturecleavage products are listed in the Examples in Table 1 including theirnoted amino acid residue sequences.

Although cortistatin-13 is unrelated to known species, cortistatin-14shares 11 of 14 residues with somatostatin-14 as discussed in Example 1.

Thus, the noted nucleotide and amino acid differences betweensomatostatin and cortistatin indicate clearly that they are the productsof separate genes.

The mouse homolog to the rat preprocortistatin cDNA has a completecoding nucleotide sequence of 427 nucleotides in length, as shown inFIG. 3 and listed in SEQ ID NO 4. The complete preprocortistatin cDNAclone presents a 327 nucleotide open reading frame (ORF) with aN-terminal signal peptide whose cleavage site is between amino acidpositions 25 and 26 corresponding to a cleavage site after nucleotideposition 99.

Translation of this mouse cDNA sequence provides a novel protein of 109amino acid residues, provisionally called mouse preprocortistatin. Theamino acid sequence of mouse preprocortistatin is listed in SEQ ID NO 4with the nucleotide sequence and in SEQ ID NO 5 alone.

Similar to the rat preprocortistatin, cleavage of the mousepreprospecies to procortistatin generates a mature protein that isprocessed at either of two tandem basic amino acid pairs, KS (lys-ser)and KK (lys-lys), to produce mouse cortistatin-29 and mousecortistatin-14. As with the rat cleavage patterns, two smaller mousecortistatin species of 13 and 14 amino acid residues are produced whencleavage occurs at both sets of basic residues. The mouse preprospeciesalong with the prospecies and the mature proteins are listed in Table 1in the Examples including their noted amino acid residue sequences.

By introducing two gaps, the mouse and rat nucleotide sequences are 86%identical. Assuming that the mouse translation initiation product beginsat the second methionine triplet, it contains 108 amino acids comparedto 112 for rat. Again, after introduction of two gaps, the rat and mouseproteins share 82% identity. The mouse nucleotide sequence correspondingto cortistatin-14 and the adjacent lysine doublet that serves as itssite of proteolytic release from its precursor were identical to sameregion in the rat sequence, thus supporting a functional conservation ofthe mature peptide. The DNA sequence upstream from the processing siteof cortistatin 14 showed several points of divergence, including someresulting in non-conservative amino acid substitutions.

In view of the conserved domains and cleavage sites for generatingmature cortistatin proteins for two mammals, rats and mice, similarcleavage patterns and resultant protein species are identifiable inother mammals including humans.

The human homolog to the rat preprocortistatin cDNA has a completecoding nucleotide sequence of 701 nucleotides in length, as shown inFIG. 3a and listed in SEQ ID NO 25. The complete preprocortistatin cDNAclone presents a 315 nucleotide open reading frame, beginning atposition 78 of SEQ ID NO 25.

Translation of this human cDNA sequence provides a novel protein of 105amino acid residues, provisionally called human preprocortistatin. Theamino acid sequence of human preprocortistatin is shown in FIG. 3b, andlisted in SEQ ID NO 26.

Similar to the rat preprocortistatin, cleavage of the humanpreprospecies to procortistatin generates a mature protein that isprocessed at either of two RR (arg-arg) tandem basic amino acid pairs,to produce human cortistatin-29 (positions 77 to 105 of SEQ ID NO. 26)and human cortistatin-17 (positions 89 to 105 of SEQ ID NO 26). Thehuman preprospecies along with the prospecies and the mature proteinsare listed in Table 1 in the Examples including their noted amino acidresidue sequences.

The human and rat nucleotide sequences are 71% identical. The humancortistatin-17 shares 13 of the last 14 residues with rat and mousecortistatin-14. The lysine doublet that lies just N-terminal tocortistatin-14 in the rat and mouse is not conserved in the humansequence. The DNA sequence upstream from the processing site ofcortistatin-14 are not very conserved across species. However, ratcortistatin-31 and human cortistatin-31 (positions 44 to 74 of SEQ ID NO26) share 13 residues clustered in their N-terminal regions that areconserved among the rat, mouse, and human prohormone sequences.

A cortistatin protein of this invention can be in a variety of forms,depending upon the use therefor, as described herein. For example, acortistatin can be isolated from a natural tissue.

Alternatively, a cortistatin of this invention can be recombinantprotein, that is, produced by recombinant DNA (rDNA) methods asdescribed herein. A recombinant cortistatin protein need not necessarilybe substantially pure, or even isolated, to be useful in certainembodiments, although recombinant production methods are a preferredmeans to produce a source for further purification to yield an isolatedor substantially pure receptor composition. A recombinant cortistatinprotein can be present in or on a mammalian cell line or in crudeextracts of a mammalian cell line.

In one embodiment, a cortistatin protein is substantially free of otherneuropeptides, so that the purity of a cortistatin reagent and freedomfrom pharmacologically distinct proteins affords use in the screeningmethods. The recombinant production methods are ideally suited toproduce absolute purity in this regard, although biochemicalpurification methods from natural sources are also contemplated. In thisregard, a cortistatin protein is substantially free from otherneuropeptides if there are insufficient other neuropeptides such thatpharmacological cross-reactivity is not detected in conventionalscreening assays for ligand binding or biological activity.

Preferably, a cortistatin protein of this invention is present in acomposition in an isolated form, i.e., comprising at least about 0.1percent by weight of the total composition, preferably at least 1%, andmore preferably at least about 90%. Particularly preferred is asubstantially pure preparation of cortistatin, that is at least 90% byweight, and more preferably at least 99% by weight. Biochemical methodsuseful for the enrichment and preparation of an isolated cortistatinbased on the chemical properties of a polypeptide are well known, andcan be routinely used for the production of proteins which are enrichedby greater than 99% by weight.

An isolated or recombinant cortistatin protein of this invention can beused for a variety of purposes, as described further herein. Acortistatin protein can be used as an immunogen to produce antibodiesimmunoreactive with cortistatin. Cortistatin proteins can be used in invitro ligand binding assays for identifying ligand bindingspecificities, and agonists or antagonists thereto, to characterizecandidate pharmaceutical compounds useful for modulating cortistatinfunction, and as therapeutic agents for effecting cortistatin functions.Other uses will be readily apparent to one skilled in the art.

Furthermore, the invention contemplates analogs of a cortistatin proteinof this invention. An analog is a man-made variant which exhibits thequalities of a cortistatin of this invention in terms of immunologicalreactivity, ligand binding capacity or the like functional properties ofa cortistatin protein of this invention. An analog can therefore be acleavage product of cortistatin, can be a polypeptide corresponding to aportion of cortistatin, can be cortistatin polypeptide in which amembrane anchor has been removed, and can be a variant cortistatinsequence in which some amino acid residues have been altered, to name afew permutations.

Insofar as the present disclosure identifies cortistatin from differentmammalian species, the present invention is not to be limited to acortistatin protein derived from one or a few mammalian species. Thus,the invention contemplates a mammalian cortistatin protein, which can bederived, by rDNA or biochemical purification from natural sources, fromany of a variety of species including man, mouse, rabbit, rat, dog, cat,sheep, cow, and the like mammalian species, without limitation. Humanand agriculturally relevant animal species are particularly preferred.

Exemplary cortistatin species identified herein are mouse, rat and humancortistatin.

The nucleotide (cDNA) sequence of rat preprocortistatin is shown in SEQID NO 1, and corresponding amino acid residue sequence of ratpreprocortistatin is shown in SEQ ID NO 2. The amino acid residuesequence of rat procortistatin is shown in SEQ ID NO 6, and cleavageproducts are shown in SEQ ID NOs 7, 8 and 9.

The nucleotide (cDNA) sequence of mouse preprocortistatin is shown inSEQ ID NO 4, and corresponding amino acid residue sequence of mousepreprocortistatin is shown in SEQ ID NO 5. The amino acid residuesequence of mouse procortistatin is shown in SEQ ID NO 10, and cleavageproducts are shown in SEQ ID NOs 8, 11 and 12.

The nucleotide (cDNA) sequence of human preprocortistatin is shown inSEQ ID NO 25, and corresponding amino acid residue sequence of humanpreprocortistatin is shown in SEQ ID NO 26. The amino acid residuesequences of the cleavage products are located at positions 44 to 74(human cortistatin-31), positions 77 to 105 (human cortistatin-29), andpositions 89 to 105 (human cortistatin-17) of SEQ ID NO 26. The peptidedesignated rat cortistatin-14 is highly conserved among species, and isidentical in sequence between mouse and rat and shares 13 of the last 14residues with human cortistatin-17. The amino acid residue sequence ofmouse and rat cortistatin-14 is shown in SEQ ID NO 8, and the amino acidresidue sequence of human cortistatin-17 is shown in positions 89 to 105of SEQ ID NO 26.

A cortistatin protein of this invention can be prepared by a variety ofmeans, although expression in a mammalian cell using a rDNA expressionvector is preferred. Exemplary production methods for a recombinantcortistatin are described in the Examples.

Thus, the invention also provides a method for the production ofisolated cortistatin proteins, either as intact cortistatin protein, asfusion proteins or as smaller polypeptide fragments of cortistatin. Theproduction method generally involves inducing cells to express acortistatin protein of this invention, recovering the cortistatin fromthe resulting cells, and purifying the cortistatin so recovered bybiochemical fractionation methods, using a specific antibody of thisinvention, or other chemical procedures.

The inducing step can comprise inserting a rDNA vector encoding acortistatin protein, or fragment thereof, of this invention, which rDNAis capable of expressing a cortistatin, into a suitable host cell, andexpressing the vector's cortistatin gene.

As used herein, the phrase “cortistatin polypeptide” refers to apolypeptide having an amino acid residue sequence that comprises anamino acid residue sequence that corresponds, and preferably isidentical, to a portion of a cortistatin of this invention.

A cortistatin polypeptide of the present invention has a variety of usesaccording to the present invention.

Thus, a cortistatin polypeptide of this invention is characterized byits ability to immunologically mimic an epitope (antigenic determinant)expressed by a cortistatin of this invention. Such a polypeptide isuseful herein as a component in an inoculum for producing antibodiesthat immunoreact with native cortistatin and as an antigen inimmunologic methods. Representative and preferred cortistatinpolypeptides for use as an immunogen in an inoculum are shown herein.

As used herein, the phrase “immunologically mimic” in its variousgrammatical forms refers to the ability of a cortistatin polypeptide ofthis invention to immunoreact with an antibody of the present inventionthat recognizes a conserved native epitope of a cortistatin as definedherein.

It should be understood that a subject polypeptide need not be identicalto the amino acid residue sequence of a cortistatin receptor, so long asit includes the required sequence.

In addition, certain cortistatin polypeptides derived from receptorbinding portions of cortistatin have the capacity to inhibit the bindingof the cortistatin that would normally bind a cortistatin receptor.Thus, the invention also contemplates cortistatin polypeptides which arespecifically designed for their capacity to mimic exposed regions ofcortistatin involved in cortistatin receptor binding interactions andthereby receptor function. Therefore, these polypeptides have thecapacity to function as analogs to cortistatin, and thereby blockfunction. Such inhibitors of binding are referred to as therapeuticpolypeptides because of their inhibitory capacity.

In addition, polypeptides corresponding to exposed domains have theability to induce antibody molecules that immunoreact with a cortistatinof this invention at portions of cortistatin involved in receptorprotein function, and therefor the antibodies are also useful atmodulating normal cortistatin function.

A cortistatin polypeptide is preferably no more than about 120 aminoacid residues in length for reasons of ease of synthesis. Thus, it morepreferred that a cortistatin polypeptide be no more that about 100 aminoacid residues, still more preferably no more than about 50 residues, andmost preferably less than 30 amino acid residues in length whensynthetic methods of production are used.

Thus, the present invention also contemplates a cortistatin polypeptidethat has an amino acid residue sequence that corresponds to the sequenceof the cortistatin protein shown in the sequence listings, and includesan amino acid residue sequence represented by a formula selected fromthe group consisting of the polypeptides shown in the sequence listings.In this embodiment, the polypeptide is further characterized as havingthe ability to mimic a cortistatin epitope and thereby inhibitscortistatin function in a classic cortistatin receptor activation assay,as described herein.

Due to the three dimensional structure of a native folded cortistatinmolecule, the present invention contemplates that multiple regions ofcortistatin are involved in cortistatin receptor function, whichmultiple and various regions are defined by the various cortistatinpolypeptides described above. The ability of the above-describedpolypeptides to inhibit receptor-ligand binding can readily be measuredin a ligand binding assay as is shown in the Examples herein. Similarly,the ability of the above-described polypeptides to inhibit cortistatinreceptor function can readily be measured in a receptor assay as isdescribed herein.

Thus, in another embodiment, the invention contemplates cortistatinpolypeptide compositions that comprise one or more of the differentcortistatin polypeptides described above which inhibit cortistatinreceptor function, admixed in combinations to provide simultaneousinhibition of multiple contact sites on the cortistatin receptor.

A subject polypeptide includes any analog, fragment or chemicalderivative of a polypeptide whose amino acid residue sequence is shownherein so long as the polypeptide is capable of mimicking an epitope ofcortistatin. Therefore, a present polypeptide can be subject to variouschanges, substitutions, insertions, and deletions where such changesprovide for certain advantages in its use. In this regard, a cortistatinpolypeptide of this invention corresponds to, rather than is identicalto, the sequence of a cortistatin protein where one or more changes aremade and it retains the ability to induce antibodies that immunoreactwith a cortistatin of this invention.

The term “analog” includes any polypeptide having an amino acid residuesequence substantially identical to a sequence specifically shown hereinin which one or more residues have been conservatively substituted witha functionally similar residue and which displays the ability to induceantibody production as described herein. Examples of conservativesubstitutions include the substitution of one non-polar (hydrophobic)residue such as isoleucine, valine, leucine or methionine for another,the substitution of one polar (hydrophilic) residue for another such asbetween arginine and lysine, between glutamine and asparagine, betweenglycine and serine, the substitution of one basic residue such aslysine, arginine or histidine for another, or the substitution of oneacidic residue, such as aspartic acid or glutamic acid for another.

The phrase “conservative substitution” also includes the use of achemically derivatized residue in place of a non-derivatized residueprovided that such polypeptide displays the requisite binding activity.

“Chemical derivative” refers to a subject polypeptide having one or moreresidues chemically derivatized by reaction of a functional side group.Such derivatized molecules include for example, those molecules in whichfree amino groups have been derivatized to form amine hydrochlorides,p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonylgroups, chloroacetyl groups or formyl groups. Free carboxyl groups maybe derivatized to form salts, methyl and ethyl esters or other types ofesters or hydrazides. Free hydroxyl groups may be derivatized to formO-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine maybe derivatized to form N-im-benzylhistidine. Also included as chemicalderivatives are those peptides which contain one or more naturallyoccurring amino acid derivatives of the twenty standard amino acids. Forexamples: 4-hydroxyproline may be substituted for proline;5-hydroxylysine may be substituted for lysine; 3-methylhistidine may besubstituted for histidine; homoserine may be substituted for serine; andornithine may be substituted for lysine. D-amino acids may also beincluded in place of one or more L-amino acids. Polypeptides of thepresent invention also include any polypeptide having one or moreadditions and/or deletions or residues relative to the sequence of apolypeptide whose sequence is shown herein, so long as the requisiteactivity is maintained.

The term “fragment” refers to any subject polypeptide having an aminoacid residue sequence shorter than that of a polypeptide whose aminoacid residue sequence is shown herein.

When a polypeptide of the present invention has a sequence that is notidentical to the sequence of a cortistatin polypeptide, it is typicallybecause one or more conservative or non-conservative substitutions havebeen made, usually no more than about 30 number percent, more usually nomore than 20 number percent, and preferably no more than 10 numberpercent of the amino acid residues are substituted. Additional residuesmay also be added at either terminus for the purpose of providing a“linker” by which the polypeptides of this invention can be convenientlyaffixed to a label or solid matrix, or carrier. Preferably the linkerresidues do not form a cortistatin epitope, i.e., are not similar isstructure to a cortistatin protein.

Labels, solid matrices and carriers that can be used with thepolypeptides of this invention are described hereinbelow.

Amino acid residue linkers are usually at least one residue and can be40 or more residues, more often 1 to 10 residues, but do not form acortistatin epitope. Typical amino acid residues used for linking aretyrosine, cysteine, lysine, glutamic and aspartic acid, or the like. Inaddition, a subject polypeptide can differ, unless otherwise specified,from the natural sequence of a cortistatin protein by the sequence beingmodified by terminal-NH₂ acylation, e.g., acetylation, or thioglycolicacid amidation, by terminal-carboxlyamidation, e.g., with ammonia,methylamine, and the like.

When coupled to a carrier to form what is known in the art as acarrier-hapten conjugate, a cortistatin polypeptide of the presentinvention is capable of inducing antibodies that immunoreact withcortistatin. In view of the well established principle of immunologiccross-reactivity, the present invention therefore contemplatesantigenically related variants of the polypeptides shown herein. An“antigenically related variant” is a subject polypeptide that is capableof inducing antibody molecules that immunoreact with a polypeptidedescribed herein and with a cortistatin protein of this invention.

Any peptide of the present invention may be used in the form of apharmaceutically acceptable salt. Suitable acids which are capable offorming salts with the peptides of the present invention includeinorganic acids such as hydrochloric acid, hydrobromic acid, perchloricacid, nitric acid, thiocyanic acid, sulfuric acid, phosphoric aceticacid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalicacid, malonic acid, succinic acid, maleic acid, fumaric acid,anthranilic acid, cinnamic acid, naphthalene sulfonic acid, sulfanilicacid or the like.

Suitable bases capable of forming salts with the peptides of the presentinvention include inorganic bases such as sodium hydroxide, ammoniumhydroxide, potassium hydroxide and the like; and organic bases such asmono-, di- and tri-alkyl and aryl amines (e.g. triethylamine,diisopropyl amine, methyl amine, dimethyl amine and the like) andoptionally substituted ethanolamines (e.g. ethanolamine, diethanolamineand the like).

A cortistatin polypeptide of the present invention, also referred toherein as a subject polypeptide, can be synthesized by any of thetechniques that are known to those skilled in the polypeptide art,including recombinant DNA techniques. Synthetic chemistry techniques,such as a solid-phase Merrifield-type synthesis, are preferred forreasons of purity, antigenic specificity, freedom from undesired sideproducts, ease of production and the like. An excellent summary of themany techniques available can be found in J. M. Steward and J. D. Young,“Solid Phase Peptide Synthesis”, W. H. Freeman Co., San Francisco, 1969;M. Bodanszky, et al., “Peptide Synthesis”, John Wiley & Sons, SecondEdition, 1976 and J. Meienhofer, “Hormonal Proteins and Peptides”, Vol.2, p. 46, Academic Press (New York), 1983 for solid phase peptidesynthesis, and E. Schroder and K. Kubke, “The Peptides”, Vol. 1,Academic Press (New York), 1965 for classical solution synthesis, eachof which is incorporated herein by reference. Additional peptidesynthesis methods are described by Sutcliffe in U.S. Pat. No. 4,900,811and 5,242,798, which are hereby incorporated by reference. Appropriateprotective groups usable in such synthesis are described in the abovetexts and in J. F. W. McOmie, “Protective Groups in Organic Chemistry”,Plenum Press, New York, 1973, which is incorporated herein by reference.

In general, the solid-phase synthesis methods contemplated comprise thesequential addition of one or more amino acid residues or suitablyprotected amino acid residues to a growing peptide chain. Normally,either the amino or carboxyl group of the first amino acid residue isprotected by a suitable, selectively removable protecting group. Adifferent, selectively removable protecting group is utilized-for aminoacids containing a reactive side group such as lysine.

Using a solid phase synthesis as exemplary, the protected or derivatizedamino acid is attached to an inert solid support through its unprotectedcarboxyl or amino group. The protecting group of the amino or carboxylgroup is then selectively removed and the next amino acid in thesequence having the complimentary (amino or carboxyl) group suitablyprotected is admixed and reacted under conditions suitable for formingthe amide linkage with the residue already attached to the solidsupport. The protecting group of the amino or carboxyl group is thenremoved from this newly added amino acid residue, and the next aminoacid (suitably protected) is then added, and so forth. After all thedesired amino acids have been linked in the proper sequence, anyremaining terminal and side group protecting groups (and solid support)are removed sequentially or concurrently, to afford the finalpolypeptide.

A cortistatin polypeptide can be used, inter alia, in the diagnosticmethods and systems of the present invention to detect a cortistatinreceptor or cortistatin itself present in a body sample, or can be usedto prepare an inoculum as described herein for the preparation ofantibodies that immunoreact with conserved epitopes on cortistatin.

In addition, certain of the cortistatin polypeptides of this inventioncan be used in the therapeutic methods of the present invention toinhibit cortistatin function as described further herein.

C. Nucleic Acids and Polynucleotides

The DNA segments of the present invention are characterized as includinga DNA sequence that encodes a cortistatin protein of this invention.That is, the DNA segments of the present invention are characterized bythe presence of some or all of a cortistatin structural gene. Preferablythe gene is present as an uninterrupted linear series of codons whereeach codon codes for an amino acid residue found in the cortistatinprotein, i.e., a gene free of introns.

One preferred embodiment is a DNA segment that codes an amino acidresidue sequence that defines a cortistatin protein as defined herein,and the DNA segment is capable of expressing a cortistatin protein ofthis invention. A preferred DNA segment codes for an amino acid residuesequence substantially the same as, and preferably consistingessentially of, an amino acid residue sequence shown in the sequencelisting for a cortistatin protein, such as in SEQ ID NOs 2, 5, 6, 7, 8,9, 10, 11, 12, 26, positions 44 to 74 of SEQ ID NO 26, positions 77 to105 of SEQ ID NO 26, and positions 89 to 105 of SEQ ID NO 26.Particularly preferred DNA segments have a nucleotide sequence derivedfrom the sequence shown in SEQ ID NOs 1 ,4 or 25. Representative andpreferred DNA segments are described in the Examples.

Homologous DNA and RNA sequences that encode the above cortistatinprotein are also contemplated.

The amino acid residue sequence of a protein or polypeptide is directlyrelated via the genetic code to the deoxyribonucleic acid (DNA) sequenceof the structural gene that codes for the protein. Thus, a structuralgene or DNA segment can be defined in terms of the amino acid residuesequence, i.e., protein or polypeptide, for which it codes.

An important and well known feature of the genetic code is itsredundancy. That is, for most of the amino acids used to make proteins,more than one coding nucleotide triplet (codon) can code for ordesignate a particular amino acid residue. Therefore, a number ofdifferent nucleotide sequences may code for a particular amino acidresidue sequence. Such nucleotide sequences are considered functionallyequivalent since they can result in the production of the same aminoacid residue sequence in all organisms. occasionally, a methylatedvariant of a purine or pyrimidine may be incorporated into a givennucleotide sequence. However, such methylations do not affect the codingrelationship in any way.

A nucleic acid is any polynucleotide or nucleic acid fragment, whetherit be a polyribonucleotide of polydeoxyribonucleotide, i.e., RNA or DNA,or analogs thereof. In preferred embodiments, a nucleic acid molecule isin the form of a segment of duplex DNA, i.e, a DNA segment, although forcertain molecular biological methodologies, single-stranded DNA or RNAis preferred.

DNA segments (i.e., synthetic oligonucleotides) that encode portions ofcortistatin proteins can easily be synthesized by chemical techniques,for example, the phosphotriester method of Matteucci, et al., (J. Am.Chem. Soc., 103:3185-3191, 1981) or using automated synthesis methods.In addition, larger DNA segments can readily be prepared by well knownmethods, such as synthesis of a group of oligonucleotides that definethe DNA segment, followed by hybridization and ligation ofoligonucleotides to build the complete segment.

Of course, by chemically synthesizing the coding sequence, any desiredmodifications can be made simply by substituting the appropriate basesfor those encoding the native amino acid residue sequence.

Furthermore, DNA segments consisting essentially of structural genesencoding a cortistatin protein can be obtained from recombinant DNAmolecules containing a gene that defines a cortistatin protein of thisinvention, and can be subsequently modified, as by site directedmutagenesis, to introduce any desired substitutions.

1. Cloning Cortistatin Genes

Cortistatin genes of this invention can be cloned by a variety ofcloning methods and from any mammalian species. The cloning is based onthe observation that there is a significant degree of homology betweenmammalian species for any given cortistatin of this invention, andtherefor can be conducted according to the general methods described inthe Examples, using nucleic acid homology strategies.

A typical degree of homology required to successfully clone acortistatin is at least about 80% homologous at the DNA level, and atleast about 90% homologous at the protein level. Preferred cloningstrategies for isolating a nucleic acid molecule that encodes acortistatin molecule of this invention are described in the Examples,and includes the recitation of polynucleotide probes useful for thescreening of libraries of nucleic acid molecules believed to contain atarget cortistatin gene. Particularly preferred probes encode theconserved region defined by “cortistatin-14” as described herein.

Sources of libraries for cloning a cortistatin gene of this inventioncan include genomic DNA or messenger RNA (mRNA) in the form of a cDNAlibrary from a tissue believed to express a cortistatin of thisinvention. Preferred tissues are brain tissues, particularly cerebralcortex or hippocampal tissue.

The similarities between rat and mouse cortistatin are further extendedto the identification of a sequence of iteration of trinucleotide CTGrepeats. For the rat, a sequence of six iterations of the trinucleotideCTG repeats is present encoding leucine residues. In the mouse, asequence of three iterations of the trinucleotide CTG is present, alsowithin the region encoding the signal sequence. Thus, the presence ofthe iterations is typically located within the coding region for thesignal peptide.

Such a triplet expansion in other genes has been implicated as causal inneurological diseases, e.g., myotonic dystrophy as described by Brook etal., Cell, 68:799-808 (1992) and fragile-X syndrome as described by Fuet al., Cell, 67:1047-1058 (1991). In myotonic dystrophy patients whoare mildly affected, at least 50 CTG repeats are present. In severelyaffected individuals, the expansion can exist up to several kilobasepairs. In contrast, in the normal population, the repeat sequence ishighly variable ranging from 5 to 27 copies. Individuals with varyingseverities of fragile-X have been similarly characterized.

Thus, screening for the presence of a region of DNA in which the repeatsare present in either normal, underexpansion or overexpansion form canprovide a genetic basis for diagnosis for some diseases. The same may betrue for cortistatin in that expansion of the region may contribute tothe basis for a sleep-related or neuronal depressant-related disorder ordisease of the brain.

That the mouse iteration sequence is shorter than that of the rat mayindicate that the iteration sequence is unstable and subject toexpansion as has been seen with other disease states.

2. Oligonucleotides

The invention also contemplates oligonucleotides useful for methods todetect the presence of a cortistatin gene or gene transcript (mRNA) in atissue by diagnostic detection methods based on the specificity ofnucleic acid hybridization or primer extension reactions.

Thus, in one embodiment, any polynucleotide probe having a sequence of aportion of a cortistatin gene of this invention, or a related andspecific sequence, is contemplated.

Hybridization probes can be of a variety of lengths from about 10 to5000 nucleotides long, although they will typically be about 20 to 500nucleotides in length. Hybridization methods are extremely well known inthe art and will not be described further here.

In a related embodiment, detection of cortistatin genes can be conductedby primer extension reactions such as the polymerase chain reaction(PCR). To that end, PCR primers are utilized in pairs, as is well known,based on the nucleotide sequence of the gene to be detected.

Particularly preferred PCR primers can be derived from any portion of acortistatin DNA sequence, but are preferentially from regions which arenot conserved in other cellular proteins.

A preferred PCR primer pair useful for detecting cortistatin genes andcortistatin gene expression are described in the Examples. Nucleotideprimers from the corresponding region of cortistatin described hereinare readily prepared and used as PCR primers for detection of thepresence or expression of the corresponding gene in any of a variety oftissues.

3. Expression Vectors

In addition, the invention contemplates a recombinant DNA molecule(rDNA) containing a DNA segment of this invention encoding a cortistatinprotein as described herein. A rDNA can be produced by operativelylinking a vector to a DNA segment of the present invention.

As used herein, the term “vector” refers to a DNA molecule capable ofautonomous replication in a cell and to which another DNA segment can beoperatively linked so as to bring about replication of the attachedsegment. A vector adapted for expression of a gene product and capableof directing the expression of a cortistatin gene is referred to hereinas an “expression vector”. Thus, a recombinant DNA molecule is a hybridDNA molecule comprising at least two nucleotide sequences not normallyfound together in nature.

The choice of vector to which a DNA segment of the present invention isoperatively linked depends directly, as is well known in the art, on thefunctional properties desired, e.g., protein expression, and the hostcell to be transformed, these being limitations inherent in the art ofconstructing recombinant DNA molecules. However, a vector contemplatedby the present invention is at least capable of directing thereplication, and preferably also expression, of a cortistatin structuralgene included in DNA segments to which it is operatively linked.

In one embodiment, a vector contemplated by the present inventionincludes a procaryotic replicon, i.e., a DNA sequence having the abilityto direct autonomous replication and maintenance of the recombinant DNAmolecule extrachromosomally in a procaryotic host cell, such as abacterial host cell, transformed therewith. Such replicons are wellknown in the art. In addition, those embodiments that include aprocaryotic replicon also include a gene whose expression confers drugresistance to a bacterial host transformed therewith. Typical bacterialdrug resistance genes are those that confer resistance to ampicillin ortetracycline.

Those vectors that include a procaryotic replicon can also include aprocaryotic promoter capable of directing the expression (transcriptionand translation) of a cortistatin gene in a bacterial host cell, such asE. coli, transformed therewith. A promoter is an expression controlelement formed by a DNA sequence that permits binding of RNA polymeraseand transcription to occur. Promoter sequences compatible with bacterialhosts are typically provided in plasmid vectors containing convenientrestriction sites for insertion of a DNA segment of the presentinvention. Typical of such vector plasmids are pUC8, pUC9, pBR322 andpBR329 available from Biorad Laboratories, (Richmond, Calif.), pRSETavailable from Invitrogen (San Diego, Calif.) and pPL and pKK223available from Pharmacia, Piscataway, N.J.

Expression vectors compatible with eucaryotic cells, preferably thosecompatible with vertebrate cells, can also be used to form therecombinant DNA molecules of the present invention. Eucaryotic cellexpression vectors are well known in the art and are available fromseveral commercial sources. Typically, such vectors are providedcontaining convenient restriction sites for insertion of the desired DNAsegment. Typical of such vectors are PSVL and pKSV-10 (Pharmacia),pBPV-1/pML2d (International Biotechnologies, Inc.), pTDT1 (ATCC,#31255), pRc/CMV (Invitrogen, Inc.), the vector pCMV4 described herein,and the like eucaryotic expression vectors.

In preferred embodiments, the eucaryotic cell expression vectors used toconstruct the recombinant DNA molecules of the present invention includea selection marker that is effective in an eucaryotic cell, preferably adrug resistance selection marker. A preferred drug resistance marker isthe gene whose expression results in neomycin resistance, i.e., theneomycin phosphotransferase (neo) gene. Southern et al., J. Mol. Appl.Genet., 1:327-341 (1982). Alternatively, the selectable marker can bepresent on a separate plasmid, and the two vectors are introduced byco-transfection of the host cell, and selected by culturing in theappropriate drug for the selectable marker.

4. Inhibitory Nucleic Acids

In accordance with one embodiment of the invention, nucleic acidmolecules can be used in methodologies for the inhibition of cortistatingene expression, thereby inhibiting the function of thecortistatin:cortistatin receptor binding interaction by blockingcortistatin expression.

To that end, the invention contemplates isolated nucleic acid molecules,preferably single-stranded nucleic acid molecules (oligonucleotides),having a sequence complementary to a portion of a structural geneencoding a cortistatin protein of this invention. Nucleic acid-basedinhibition is well known and generally referred to as “anti-sense”technology by virtue of the use of nucleotide sequences havingcomplementarity which can hybridize to the “sense” strand or mRNA, andthereby perturb gene expression.

Typical oligonucleotides for this purpose are about 10 to 5,000,preferably about 20-1000, nucleotides in length and have a sequencecapable of hybridizing specifically with a structural protein region ofthe nucleotide sequence that encodes a cortistatin protein of thisinvention.

In one embodiment, the invention contemplates repetitive units of thenucleotide sequence complementary to a portion of a cortistatinstructural gene so as to present multiple sites for complementarybinding to the structural gene. This feature may be provided in a singlenucleic acid segment having repeating sequences defining multipleportions of a structural gene, by physical conjugation of DNA segmentseach containing a single portion of a structural gene, or a combinationthereof comprising conjugates of DNA segments, each having one or moresequences complementary to a structural gene.

It is also contemplated that nucleotide base modifications can be madeto provide certain advantages to a DNA segments of this invention,referred to as nucleotide analogs.

A nucleotide analog refers to moieties which function similarly tonucleotide sequences in a nucleic acid molecule of this invention butwhich have non-naturally occurring portions. Thus, nucleotide analogscan have altered sugar moieties or inter-sugar linkages. Exemplary arethe phosphorothioate and other sulfur-containing species, analogs havingaltered base units, or other modifications consistent with the spirit ofthis invention.

Preferred modifications include, but are not limited to, the ethyl ormethyl phosphonate modifications disclosed in U.S. Pat. No. 4,469,863and the phosphorothioate modified deoxyribonucleotides described byLaPlanche et al., Nucl. Acids Res., 14:9081, 1986; and Stec et al., J.Am. Chem. Soc., 106:6077, 1984. These modifications provide resistanceto nucleolytic degradation, thereby contributing to the increasedhalf-life in therapeutic modalities. Preferred modifications are themodifications of the 3′-terminus using phosphothioate (PS) sulfurizationmodification described by Stein et al., Nucl. Acids Res., 16:3209, 1988.

In accordance with the methods of this invention in certain preferredembodiments, at least some of the phosphodiester bonds of the nucleotidesequence can be substituted with a structure which functions to enhancethe ability of the compositions to penetrate into the region of cellswhere the cortistatin structural gene to be inhibited is located. It ispreferred that such linkages be sulfur containing as discussed above,such as phosphorotioate bonds. Other substitutions can include alkylphosphothioate bonds, N-alkyl phosphoramidates, phosphorodithioates,alkyl phosphonates, and short chain alkyl or cycloalkyl structures. Inaccordance with other preferred embodiments, the phosphodiester bondsare substituted with structures which are, at once, substantiallynon-ionic and non-chiral.

D. Anti-Cortistatin Antibodies

An antibody of the present invention, i.e., an anti-cortistatinantibody, in one embodiment is characterized as comprising antibodymolecules that immunoreact with a cortistatin protein of this invention.Preferably, an antibody further immunoreacts with a cortistatin proteinin situ, i.e., in a tissue section.

Thus, the invention describes an anti-cortistatin antibody thatimmunoreacts with any of the cortistatin polypeptides of this invention,preferably also immunoreacts with the corresponding recombinantcortistatin protein, and more preferably also reacts with a nativeprotein in situ in a tissue section. Preferably, and antibody issubstantially free from immunoreaction with a somatostatin protein orneuropeptides other than cortistatin. Assays for immunoreaction usefulfor assessing immunoreactivity are described herein.

In one embodiment, antibody molecules are described that immunoreactwith a cortistatin receptor polypeptide of the present invention andthat have the capacity to immunoreact with an exposed site oncortistatin that is required for cortistatin receptor binding. Thus,preferred antibody molecules in this embodiment also inhibit cortistatinreceptor function, and are therefore useful therapeutically to block thereceptor's function.

Exemplary cortistatin inhibitory antibodies immunoreact with acortistatin polypeptide described herein that defines an exposed regionof a cortistatin protein that is involved in cortistatin receptorfunction, such as ligand binding.

An antibody of the present invention is typically produced by immunizinga mammal with an inoculum containing a cortistatin polypeptide of thisinvention and thereby induce in the mammal antibody molecules havingimmunospecificity for immunizing polypeptide. The antibody molecules arethen collected from the mammal and isolated to the extent desired bywell known techniques such as, for example, by using DEAE Sephadex toobtain the IgG fraction. Exemplary antibody preparation methods usingcortistatin polypeptides in the immunogen are described herein in theExamples.

The term “antibody” in its various grammatical forms is used herein as acollective noun that refers to a population of immunoglobulin moleculesand/or immunologically active portions of immunoglobulin molecules,i.e., molecules that contain an antibody combining site or paratope.

An “antibody combining site” is that structural portion of an antibodymolecule comprised of heavy and light chain variable and hypervariableregions that specifically binds antigen.

The phrase “antibody molecule” in its various grammatical forms as usedherein contemplates both an intact immunoglobulin molecule and animmunologically active portion of an immunoglobulin molecule.

Exemplary antibody molecules for use in the diagnostic methods andsystems of the present invention are intact immunoglobulin molecules,substantially intact immunoglobulin molecules and those portions of animmunoglobulin molecule that contain the paratope, including thoseportions known in the art as Fab, Fab′, F(ab′)₂ and F(v).

Fab and F(ab′)₂ portions of antibodies are prepared by the proteolyticreaction of papain and pepsin, respectively, on substantially intactantibodies by methods that are well known. See for example, U.S. Pat.No. 4,342,566 to Theofilopolous and Dixon. Fab′ antibody portions arealso well known and are produced from F(ab′)₂ portions followed byreduction of the disulfide bonds linking the two heavy reduction of thedisulfide bonds linking the two heavy chain portions as withmercaptoethanol, and followed by alkylation of the resulting proteinmercaptan with a reagent such as iodoacetamide. An antibody containingintact antibody molecules are preferred, and are utilized asillustrative herein.

The preparation of antibodies against polypeptide is well known in theart. See Staudt et al., J. Exp. Med., 157:687-704 (1983), or theteachings of Sutcliffe, J. G., as described in U.S. Pat. No. 4,900,811,the teaching of which are hereby incorporated by reference.

Briefly, to produce a peptide antibody composition of this invention, alaboratory mammal is inoculated with an immunologically effective amountof a cortistatin polypeptide, typically as present in a vaccine of thepresent invention. The anti-cortistatin antibody molecules therebyinduced are then collected from the mammal and those immunospecific forboth a cortistatin polypeptide and the corresponding recombinantcortistatin protein are isolated to the extent desired by well knowntechniques such as, for example, by immunoaffinity chromatography.

To enhance the specificity of the antibody, the antibodies arepreferably purified by immunoaffinity chromatography using solidphase-affixed immunizing polypeptide. The antibody is contacted with thesolid phase-affixed immunizing polypeptide for a period of timesufficient for the polypeptide to immunoreact with the antibodymolecules to form a solid phase- affixed immunocomplex. The boundantibodies are separated from the complex by standard techniques.

The word “inoculum” in its various grammatical forms is used herein todescribe a composition containing a cortistatin polypeptide of thisinvention as an active ingredient used for the preparation of antibodiesagainst a cortistatin polypeptide. When a polypeptide is used in aninoculum to induce antibodies it is to be understood that thepolypeptide can be used in various embodiments, e.g., alone or linked toa carrier as a conjugate, or as a polypeptide polymer. However, for easeof expression and in context of a polypeptide inoculum, the variousembodiments of the polypeptides of this invention are collectivelyreferred to herein by the term “polypeptide” and its various grammaticalforms.

For a polypeptide that contains fewer than about 35 amino acid residues,it is preferable to use the peptide bound to a carrier for the purposeof inducing the production of antibodies.

One or more additional amino acid residues can be added to the amino- orcarboxy-termini of the polypeptide to assist in binding the polypeptideto a carrier. Cysteine residues added at the amino- or carboxy-terminiof the polypeptide have been found to be particularly useful for formingconjugates via disulfide bonds. However, other methods well known in theart for preparing conjugates can also be used.

The techniques of polypeptide conjugation or coupling through activatedfunctional groups presently known in the art are particularlyapplicable. See, for example, Aurameas, et al., Scand. J. Immunol., Vol.8, Suppl. 7:7-23 (1978) and U.S. Pat. Nos. 4,493,795, 3,791,932 and3,839,153. In addition, a site-directed coupling reaction can be carriedout so that any loss of activity due to polypeptide orientation aftercoupling can be minimized. See, for example, Rodwell et al., Biotech.,3:889-894 (1985), and U.S. Pat. No. 4,671,958.

Exemplary additional linking procedures include the use of Michaeladdition reaction products, di-aldehydes such as glutaraldehyde,Klipstein, et al., J. Infect. Dis., 147:318-326 (1983) and the like, orthe use of carbodiimide technology as in the use of a water-solublecarbodiimide to form amide links to the carrier. Alternatively, theheterobifunctional cross-linker SPDP (N-succinimidyl-3-(2-pyridyldithio)proprionate)) can be used to conjugate peptides, in which acarboxy-terminal cysteine has been introduced.

Useful carriers are well known in the art, and are generally proteinsthemselves. Exemplary of such carriers are keyhole limpet hemocyanin(KLH), edestin, thyroglobulin, albumins such as bovine serum albumin(BSA) or human serum albumin (HSA), red blood cells such as sheeperythrocytes (SRBC), tetanus toxoid, cholera toxoid as well as polyaminoacids such as poly D-lysine:D-glutamic acid, and the like.

The choice of carrier is more dependent upon the ultimate use of theinoculum and is based upon criteria not particularly involved in thepresent invention. For example, a carrier that does not generate anuntoward reaction in the particular animal to be inoculated should beselected.

The present inoculum contains an effective, immunogenic amount of apolypeptide of this invention, typically as a conjugate linked to acarrier. The effective amount of polypeptide per unit dose sufficient toinduce an immune response to the immunizing polypeptide depends, amongother things, on the species of animal inoculated, the body weight ofthe animal and the chosen inoculation regimen is well known in the art.Inocula typically contain polypeptide concentrations of about 10micrograms (μg) to about 500 milligrams (mg) per inoculation (dose),preferably about 50 micrograms to about 50 milligrams per dose.

The term “unit dose” as it pertains to the inocula refers to physicallydiscrete units suitable as unitary dosages for animals, each unitcontaining a predetermined quantity of active material calculated toproduce the desired immunogenic effect in association with the requireddiluent; i.e., carrier, or vehicle. The specifications for the novelunit dose of an inoculum of this invention are dictated by and aredirectly dependent on (a) the unique characteristics of the activematerial and the particular immunologic effect to be achieved, and (b)the limitations inherent in the art of compounding such active materialfor immunologic use in animals, as disclosed in detail herein, thesebeing features of the present invention.

Inocula are typically prepared from the dried solidpolypeptide-conjugate by dispersing the polypeptide-conjugate in aphysiologically tolerable (acceptable) diluent such as water, saline orphosphate-buffered saline to form an aqueous composition.

Inocula can also include an adjuvant as part of the diluent. Adjuvantssuch as complete Freund's adjuvant (CFA), incomplete Freund's adjuvant(IFA) and alum are materials well known in the art, and are availablecommercially from several sources.

The antibody so produced can be used, inter alia, in the diagnosticmethods and systems of the present invention to detect cortistatinpresent in a sample such as a tissue section or body fluid sample.Anti-cortistatin antibodies that inhibit cortistatin function can alsobe used in vivo in therapeutic methods as described herein.

A preferred anti-cortistatin antibody is a monoclonal antibody.

The phrase “monoclonal antibody” in its various grammatical forms refersto a population of antibody molecules that contain only one species ofantibody combining site capable of immunoreacting with a particularepitope. A monoclonal antibody thus typically displays a single bindingaffinity for any epitope with which it immunoreacts. A monoclonalantibody may therefore contain an antibody molecule having a pluralityof antibody combining sites, each immunospecific for a differentepitope, e.g., a bispecific monoclonal antibody.

A preferred monoclonal antibody of this invention comprises antibodymolecules that immunoreact with a cortistatin polypeptide of the presentinvention as described for the anti-cortistatin antibodies of thisinvention. More preferably, the monoclonal antibody also immunoreactswith recombinantly produced whole cortistatin protein.

A monoclonal antibody is typically composed of antibodies produced byclones of a single cell called a hybridoma that secretes (produces) onlyone kind of antibody molecule. The hybridoma cell is formed by fusing anantibody-producing cell and a myeloma or other self-perpetuating cellline. The preparation of such antibodies was first described by Kohlerand Milstein, Nature, 256:495-497 (1975), the description of which isincorporated by reference. The hybridoma supernates so prepared can bescreened for the presence of antibody molecules that immunoreact with acortistatin polypeptide, or for inhibition of cortistatin binding tocortistatin receptor as described herein.

Briefly, to form the hybridoma from which the monoclonal antibodycomposition is produced, a myeloma or other self-perpetuating cell lineis fused with lymphocytes obtained from the spleen of a mammalhyperimmunized with a cortistatin antigen, such as is present in acortistatin polypeptide of this invention. The polypeptide-inducedhybridoma technology is described by Niman et al., Proc. Natl. Acad.Sci., USA, 80:4949-4953 (1983), the description of which is incorporatedherein by reference.

It is preferred that the myeloma cell line used to prepare a hybridomabe from the same species as the lymphocytes. Typically, a mouse of thestrain 129 GlX+is the preferred mammal. Suitable mouse myelomas for usein the present invention include thehypoxanthine-aminopterin-thymidine-sensitive (HAT) cell linesP3X63-Ag8.653, and Sp2/0-Ag14 that are available from the American TypeCulture Collection, Rockville, Md. under the designations CRL 1580 andCRL 1581, respectively.

Splenocytes are typically fused with myeloma cells using polyethyleneglycol (PEG) 1500. Fused hybrids are selected by their sensitivity toHAT. Hybridomas producing a monoclonal antibody of this invention areidentified using the enzyme linked immunosorbent assay (ELISA) describedin the Examples.

A monoclonal antibody of the present invention can also be produced byinitiating a monoclonal hybridoma culture comprising a nutrient mediumcontaining a hybridoma that produces and secretes antibody molecules ofthe appropriate polypeptide specificity. The culture is maintained underconditions and for a time period sufficient for the hybridoma to secretethe antibody molecules into the medium. The antibody-containing mediumis then collected. The antibody molecules can then be further isolatedby well known techniques.

Media useful for the preparation of these compositions are both wellknown in the art and commercially available and include syntheticculture media, inbred mice and the like. An exemplary synthetic mediumis Dulbecco's Minimal Essential Medium (DMEM; Dulbecco et al., Virol.8:396 (1959)) supplemented with 4.5 gm/1 glucose, 20 mM glutamine, and20% fetal calf serum. An exemplary inbred mouse strain is the Balb/c.

Other methods of producing a monoclonal antibody, a hybridoma cell, or ahybridoma cell culture are also well known. See, for example, the methodof isolating monoclonal antibodies from an immunological repertoire asdescribed by Sastry, et al., Proc. Natl. Acad. Sci. USA, 86:5728-5732(1989); and Huse et al., Science, 246:1275-1281 (1989).

The monoclonal antibodies of this invention can be used in the samemanner as disclosed herein for antibodies of the present invention.

For example, the monoclonal antibody can be used in the therapeutic,diagnostic or in vitro methods disclosed herein where immunoreactionwith cortistatin is desired.

Also contemplated by this invention is the hybridoma cell, and culturescontaining a hybridoma cell that produce a monoclonal antibody of thisinvention.

E. Diagnostic Methods

The present invention contemplates various assay methods for determiningthe presence, and preferably amount, of cortistatin in a body samplesuch as a tissue sample, including tissue mass or tissue section, or ina biological fluid sample using a polypeptide, polyclonal antibody ormonoclonal antibody of this invention as an immunochemical reagent toform an immunoreaction product whose amount relates, either directly orindirectly, to the amount of cortistatin in the sample.

Those skilled in the art will understand that there are numerous wellknown clinical diagnostic chemistry procedures in which animmunochemical reagent of this invention can be used to form animmunoreaction product whose amount relates to the amount of cortistatinin a body sample. Thus, while exemplary assay methods are describedherein, the invention is not so limited.

For example, in view of the demonstrated property that cortistatin bindsa cortistatin receptor, a cortistatin protein of this invention can beused directly as a probe for detection of a cortistatin receptor bybinding thereto.

Additionally, one can use a nucleic acid molecule probes describedherein to detect the presence in a cell or tissue of a cortistatin geneor expressed gene in the form of mRNA encoding a cortistatin protein ofthis invention, as described further herein. Suitable probe-based assaysare described by Sutcliffe in U.S. Pat. Nos . 4,900,811 and 5,242,798,the disclosures of which are incorporated by reference.

Various heterogenous and homogeneous protocols, either competitive ornoncompetitive, can be employed in performing an assay method of thisinvention.

For example, one embodiment contemplates a method for assaying theamount of cortistatin protein in a sample that utilizes ananti-cortistatin antibody to immunoreact with cortistatin protein in asample. In this embodiment, the antibody immunoreacts with cortistatinto form a cortistatin-antibody immunoreaction complex, and the complexis detected indicating the presence of cortistatin in the sample.

An immunoassay method using an anti-cortistatin antibody molecule forassaying the amount of cortistatin in a sample typically comprises thesteps of:

(a) Forming an immunoreaction admixture by admixing (contacting) asample with an anti-cortistatin antibody of the present invention,preferably a monoclonal antibody. The sample is typically in the form ofa fixed tissue section in a solid phase such that the immunoreactionadmixture has both a liquid phase and a solid phase, and the antibodyfunctions as a detection reagent for the presence of cortistatin in thesample.

Preferably, the sample is a brain tissue sample that has been preparedfor immunohistological staining as is well known, although other tissuesamples may be adsorbed onto a solid phase, including tissue extracts orbody fluid. In that case the adsorption onto a solid phase can beconducted as described for well known Western blot procedures.

(b) The immunoreaction admixture is maintained under biological assayconditions for a predetermined time period such as about 10 minutes toabout 16-20 hours at a temperature of about 4° C. to about 45° C. that,such time being sufficient for the cortistatin present in the sample toimmunoreact with (immunologically bind) the antibody and form acortistatin-containing immunoreaction product (immunocomplex).

Biological assay conditions are those that maintain the biologicalactivity of the immunochemical reagents of this invention and thecortistatin sought to be assayed. Those conditions include a temperaturerange of about 4° C. to about 45° C., a pH value range of about 5 toabout 9 and an ionic strength varying from that of distilled water tothat of about one molar sodium chloride. Methods for optimizing suchconditions are well known in the art.

(c) The presence, and preferably amount, of cortistatin-containingimmunoreaction product that formed in step (b) is determined (detected),thereby determining the amount of cortistatin present in the sample.

Determining the presence or amount of the immunoreaction product, eitherdirectly or indirectly, can be accomplished by assay techniques wellknown in the art, and typically depend on the type of indicating meansused.

Preferably, the determining of step (c) comprises the steps of:

(I) admixing the cortistatin-containing immunoreaction product with asecond antibody to form a second (detecting) immunoreaction admixture,said second antibody molecule having the capacity to immunoreact withthe first antibody (primary) in the immunoreaction product.

Antibodies useful as the second antibody include polyclonal ormonoclonal antibody preparations raised. against the primary antibody.

(ii) maintaining said second immunoreaction admixture for a time periodsufficient for said second antibody to complex with the immunoreactionproduct and form a second immunoreaction product, and

(iii) determining the amount of second antibody present in the secondimmunoreaction product and thereby the amount of immunoreaction productformed in step (c).

In one embodiment, the second antibody is a labeled antibody (i.e.,detecting antibody) such that the label provides an indicating means todetect the presence of the second immunoreaction product formed. Thelabel is measured in the second immunoreaction product, therebyindicating the presence, and preferably amount, of second antibody inthe solid phase.

Alternatively, the amount of second antibody can be determined bypreparation of an additional reaction admixture having an indicatingmeans that specifically reacts with (binds to) the second antibody, asis well known. Exemplary are third immunoreaction admixtures with alabeled anti-immunoglobulin antibody molecule specific for the secondantibody. After third immunoreaction, the formed third immunoreactionproduct is detected through the presence of the label.

Exemplary methods involve the use of in situ immunoreaction methodsusing tissue sections, or Western blot procedures, as described bySutcliffe in U.S. Pat. No. 4,900,811.

Another embodiment is contemplated for assaying the amount oftherapeutically administered cortistatin protein or anti-cortistatinantibody in a body fluid sample such as cerebrospinal fluid (CSF),blood, plasma or serum. The method utilizes a competition reaction inwhich either a cortistatin polypeptide or an anti-cortistatin antibodymolecule of this invention is present in the solid phase as animmobilized immunochemical reagent, and the other of the two reagents ispresent in solution in the liquid phase, in the form of a labeledreagent. A fluid sample is admixed thereto to form a competitionimmunoreaction admixture, and the resulting amount of label in the solidphase is proportional, either directly or indirectly, to the amount ofcortistatin polypeptide or antibody in the fluid sample, depending uponthe format.

Thus one version of this embodiment comprises the steps of:

(a) Forming a competition immunoreaction admixture by admixing(contacting) a fluid sample with:

(1) an anti-cortistatin antibody according to this invention containingantibody molecules that immunoreact with a cortistatin protein of thisinvention, said antibody being operatively linked to a solid matrix suchthat the competition immunoreaction admixture has both a liquid phaseand a solid phase, and

(2) a polypeptide or recombinant cortistatin protein of the presentinvention that is immunoreactive with the added antibody. The admixedpolypeptide/protein in the liquid phase (labeled competing antigen) isoperatively linked to an indicating means as described herein.

(b) The competition immunoreaction admixture is then maintained for atime period sufficient for the competing antigen and the body sampleantigen present in the liquid phase to compete for immunoreaction withthe solid phase antibody. Such immunoreaction conditions are previouslydescribed, and result in the formation of an indicating means-containingimmunoreaction product comprising the labeled competing antigen in thesolid phase.

(c) The amount of indicating means present in the product formed in step(b) is then determined, thereby determining the presence, and preferablyamount, of sample antigen present in the fluid sample.

Determining the indicating means in the solid phase is then conducted bythe standard methods described herein.

A reverse version of this embodiment comprises the steps of:

(a) Forming a competition immunoreaction admixture by admixing a fluidsample with:

(1) an anti-cortistatin antibody according to the present invention; and

(2) a cortistatin polypeptide or recombinant cortistatin protein of thepresent invention (capture antigen) that is immunoreactive with theantibody and is operatively linked to a solid matrix such that thecompetition immunoreaction admixture has both a liquid phase and a solidphase.

(b) The competition immunoreaction admixture is then maintained for atime period sufficient for any cortistatin antigen or anti-cortistatinantibody in the fluid to compete with the admixed antibody molecules forimmunoreaction with the solid phase capture antigen and form anantibody-containing immunoreaction product in the solid phase.

(c) The amount of antibody present in the product formed in step (b) isthen determined, thereby determining the presence and/or amount oftarget. material in the fluid sample.

In preferred embodiments, the antibody is operatively linked to anindicating means such that the determining in step (c) comprisesdetermining the amount of indicating means present in the product formedin step (b).

Preferably, the fluid sample is provided to a competition immunoreactionadmixture as a known amount of CSF, blood, or a blood derived productsuch as serum or plasma. Further preferred are embodiments wherein theamount of immunochemical reagent in the liquid phase of theimmunoreaction admixture is an excess amount relative to the amount ofreagent in the solid phase. Typically, a parallel set of competitionimmunoreactions are established using a known amount of purifiedrecombinant cortistatin or polypeptide in a dilution series so that astandard curve can be developed, as is well known. Thus, the amount ofproduct formed in step (c) when using a fluid sample is compared to thestandard curve, thereby determining the amount of target antigen presentin the fluid.

In another embodiment, the method for assaying the amount of cortistatinin a sample utilizes a first capture antibody to capture and immobilizecortistatin in the solid phase and a second indicator antibody toindicate the presence of the captured cortistatin antigen. In thisembodiment, one antibody immunoreacts with a cortistatin protein to forma cortistatin-antibody immunoreaction complex, and the other antibody isable to immunoreact with the cortistatin while present in thecortistatin-antibody immunoreaction complex. This embodiment can bepracticed in two formats with the immobilized capture antibody beingeither of the two above-identified antibodies, and the indicatorantibody being the other of the two antibodies.

Where a antibody is in the solid phase as a capture reagent, a preferredmeans for determining the amount of solid phase reaction product is bythe use of a labeled cortistatin polypeptide, followed by the detectionmeans described herein for other labeled products in the solid phase.

Also contemplated are immunological assays capable of detecting thepresence of immunoreaction product formation without the use of a label.Such methods employ a “detection means”, which means are themselveswell-known in clinical diagnostic chemistry and constitute a part ofthis invention only insofar as they are utilized with otherwise novelpolypeptides, methods and systems. Exemplary detection means includemethods known as biosensors and include biosensing methods based ondetecting changes in the reflectivity of a surface, changes in theabsorption of an evanescent wave by optical fibers or changes in thepropagation of surface acoustical waves.

F. Diagnostic Kits

The present invention also describes a diagnostic system, preferably inkit form, for assaying for the presence of a cortistatin of thisinvention in a body sample, such brain tissue, cell suspensions ortissue sections, or body fluid samples such as CSF, blood, plasma orserum, where it is desirable to detect the presence, and preferably theamount, of a cortistatin protein in the sample according to thediagnostic methods described herein.

In a related embodiment, a nucleic acid molecule can be used as a probe(an oligonucleotide) to detect the presence of a gene or mRNA in a cellthat is diagnostic for the presence or expression of a cortistatin inthe cell. The nucleic acid molecule probes were described in detailearlier.

The diagnostic system includes, in an amount sufficient to perform atleast one assay, a subject cortistatin polypeptide, a subject antibodyor monoclonal antibody, and/or a subject nucleic acid molecule probe ofthe present invention, as a separately packaged reagent.

In another embodiment, a diagnostic system, preferably in kit form, iscontemplated for assaying for the presence of a cortistatin polypeptideor anti-cortistatin antibody in a body fluid sample such as formonitoring the fate of therapeutically administered cortistatinpolypeptide or anti-cortistatin antibody. The system includes, in anamount sufficient for at least one assay, a subject cortistatinpolypeptide and/or a subject antibody as a separately packagedimmunochemical reagent.

Instructions for use of the packaged reagent(s) are also typicallyincluded.

As used herein, the term “package” refers to a solid matrix or materialsuch as glass, plastic (e.g., polyethylene, polypropylene orpolycarbonate), paper, foil and the like capable of holding within fixedlimits a polypeptide, polyclonal antibody or monoclonal antibody of thepresent invention. Thus, for example, a package can be a glass vial usedto contain milligram quantities of a contemplated polypeptide orantibody or it can be a microtiter plate well to which microgramquantities of a contemplated polypeptide or antibody have beenoperatively affixed, i.e., linked so as to be capable of beingimmunologically bound by an antibody or antigen, respectively.

“Instructions for use” typically include a tangible expressiondescribing the reagent concentration or at least one assay methodparameter such as the relative amounts of reagent and sample to beadmixed, maintenance time periods for reagent/sample admixtures,temperature, buffer conditions and the like.

A diagnostic system of the present invention preferably also includes alabel or indicating means capable of signaling the formation of animmunocomplex containing a polypeptide or antibody molecule of thepresent invention.

The word “complex” as used herein refers to the product of a specificbinding reaction such as an antibody-antigen or receptor-ligandreaction. Exemplary complexes are immunoreaction products.

As used herein, the terms “label” and “indicating means” in theirvarious grammatical forms refer to single atoms and molecules that areeither directly or indirectly involved in the production of a detectablesignal to indicate the presence of a complex. Any label or indicatingmeans can be linked to or incorporated in an expressed protein,polypeptide, or antibody molecule that is part of an antibody ormonoclonal antibody composition of the present invention, or usedseparately, and those atoms or molecules can be used alone or inconjunction with additional reagents. Such labels are themselveswell-known in clinical diagnostic chemistry and constitute a part ofthis invention only insofar as they are utilized with otherwise novelproteins methods and/or systems.

The labeling means can be a fluorescent labeling agent that chemicallybinds to antibodies or antigens without denaturing them to form afluorochrome (dye) that is a useful immunofluorescent tracer. Suitablefluorescent labeling agents are fluorochromes such as fluoresceinisocyanate (FIC), fluorescein isothiocyante (FITC),5-dimethylamine-1-naphthalenesulfonyl chloride (DANSC),tetramethylrhodamine isothiocyanate (TRITC), lissamine, rhodamine 8200sulphonyl chloride (RB 200 SC) and the like. A description ofimmunofluorescence analysis techniques is found in DeLuca,“Immunofluorescence Analysis”, in Antibody As a Tool, Marchalonis, etal., eds., John Wiley & Sons, Ltd., pp. 189-231 (1982), which isincorporated herein by reference.

In preferred embodiments, the indicating group is an enzyme, such ashorseradish peroxidase (HRP), glucose oxidase, or the like. In suchcases where the principal indicating group is an enzyme such as HRP orglucose oxidase, additional reagents are required to visualize the factthat a receptor-ligand complex (immunoreactant) has formed. Suchadditional reagents for HRP include hydrogen peroxide and an oxidationdye precursor such as diaminobenzidine. An additional reagent usefulwith glucose oxidase is 2,2′-amino-di-(3-ethyl-benzthiazoline-G-sulfonicacid) (ABTS).

Radioactive elements are also useful labeling agents and are usedillustratively herein. An exemplary radiolabeling agent is a radioactiveelement that produces gamma ray emissions. Elements which themselvesemit gamma rays, such as ¹²⁴I, ¹²⁵I, ¹²⁸I, ¹³²I and ⁵¹Cr represent oneclass of gamma ray emission- producing radioactive element indicatinggroups. Particularly preferred is ¹²⁵I. Another group of useful labelingmeans are those elements such as ¹¹C, ¹⁸F, ¹⁵O and ¹³N which themselvesemit positrons. The positrons so emitted produce gamma rays uponencounters with electrons present in the animal's body. Also useful is abeta emitter, such ¹¹¹ indium or ³H.

The linking of labels, i.e., labeling of, polypeptides and proteins iswell known in the art. For instance, antibody molecules produced by ahybridoma can be labeled by metabolic incorporation ofradioisotope-containing amino acids provided as a component in theculture medium. See, for example, Galfre et al., Meth. Enzymol., 73:3-46(1981). The techniques of protein conjugation or coupling throughactivated functional groups are particularly applicable. See, forexample, Aurameas, et al., Scand. J. Immunol., Vol. 8 Suppl. 7:7-23(1978), Rodwell et al., Biotech., 3:889-894 (1984), and U.S. Pat. No.4,493,795.

The diagnostic systems can also include, preferably as a separatepackage, a specific binding agent. A “specific binding agent” is amolecular entity capable of selectively binding a reagent species of thepresent invention or a complex containing such a species, but is notitself a polypeptide or antibody molecule composition of the presentinvention. Exemplary specific binding agents are second antibodymolecules, complement proteins or fragments thereof, S. aureus proteinA, and the like. Preferably the specific binding agent binds the reagentspecies when that species is present as part of a complex.

In preferred embodiments, the specific binding agent is labeled.However, when the diagnostic system includes a specific binding agentthat is not labeled, the agent is typically used as an amplifying meansor reagent. In these embodiments, the labeled specific binding agent iscapable of specifically binding the amplifying means when the amplifyingmeans is bound to a reagent species-containing complex.

The diagnostic kits of the present invention can be used in an “ELISA”format to detect the quantity of cortistatin in a sample. “ELISA” refersto an enzyme-linked immunosorbent assay that employs an antibody orantigen bound to a solid phase and an enzyme-antigen or enzyme-antibodyconjugate to detect and quantify the amount of an antigen present in asample. A description of the ELISA technique is found in Chapter 22 ofthe 4th Edition of Basic and Clinical Immunology by D. P. Sites et al.,published by Lange Medical Publications of Los Altos, Calif. in 1982 andin U.S. Pat. Nos. 3,654,090; 3,850,752; and 4,016,043, which are allincorporated herein by reference.

Thus, in some embodiments, a cortistatin polypeptide, an antibody or amonoclonal antibody of the present invention can be affixed to a solidmatrix to form a solid support that comprises a package in the subjectdiagnostic systems.

A reagent is typically affixed to a solid matrix by adsorption from anaqueous medium although other modes of affixation applicable to proteinsand polypeptides can be used that are well known to those skilled in theart. Exemplary adsorption methods are described herein.

Useful solid matrices are also well known in the art. Such materials arewater insoluble and include the cross-linked dextran available under thetrademark SEPHADEX from Pharmacia Fine Chemicals (Piscataway, N.J.);agarose; beads of polystyrene beads about 1 micron (μ) to about 5millimeters (mm) in diameter available from Abbott Laboratories of NorthChicago, Ill.; polyvinyl chloride, polystyrene, cross-linkedpolyacrylamide, nitrocellulose- or nylon-based webs such as sheets,strips or paddles; or tubes, plates or the wells of a microtiter platesuch as those made from polystyrene or polyvinylchloride.

The reagent species, labeled specific binding agent or amplifyingreagent of any diagnostic system described herein can be provided insolution, as a liquid dispersion or as a substantially dry power, e.g.,in lyophilized form. Where the indicating means is an enzyme, theenzyme's substrate can also be provided in a separate package of asystem. A solid support such as the before-described microtiter plateand one or more buffers can also be included as separately packagedelements in this diagnostic assay system.

The packaging materials discussed herein in relation to diagnosticsystems are those customarily utilized in diagnostic systems.

G. Cell Lines Expressing Cortistatin

The invention also contemplates a host cell transformed with arecombinant DNA (rDNA) molecule of the present invention. The host cellcan be either procaryotic or eucaryotic, although eucaryotic cells arepreferred, particularly mammalian cells. Preferred cells are isolated,that is, substantially homogeneous and therefor free from other celltypes or other cells having a cortistatin protein expressed therein.

A cell expressing a cortistatin of this invention has a variety of usesaccording to this invention. Particularly preferred are uses for bulkproduction of cortistatin, for the purpose of providing immunogen forproduction of antibody, for supply of therapeutic protein, for directbinding or for screening pharmaceutical compound banks for the presenceof cortistatin receptor-specific ligands, i.e., in drug screening assaysas described herein. Thus, particularly preferred are cells containing arDNA molecule that expresses a cortistatin protein of this invention.

In one embodiment, a cell is produced for transplantation into a bodytissue, thereby expressing cortistatin and providing replacementtherapy. The cell can be syngeneic, and typically will be a braintissue-derived cell, such as a hippocampal cell, neonatal brain tissuecell, glioma and the like neuronal tissue cell. Transplantation isaccomplished using surgical procedures available to a neurosurgeon wherethe transplantation is to be made into the brain, brain stem or otherneurological tissues. In preferred embodiments, the cell contains avector for expressing the cortistatin in which the expression means isunder the control of a regulatable promoter, as is well known, such thatexpression of cortistatin can be regulated.

Eucaryotic cells useful for expression of a cortistatin protein are notlimited, so long as the cell or cell line is compatible with cellculture methods and compatible with the propagation of the expressionvector and expression of the cortistatin protein gene product. Preferredeucaryotic host cells include yeast and mammalian cells, preferablyvertebrate cells such as those from a mouse, rat, monkey or humanfibroblastic cell line. Preferred eucaryotic host cells include Chinesehamster ovary (CHO) cells available from the ATCC as CCL61, NIH Swissmouse embryo cells NIH/3T3 (ATCC CRL 1658),HELA cells (ATCC CCL 2), babyhamster kidney cells (BHK), COS-7, COS-1, HEK293 (ATCC CRL 1573), Ltk-1,AV-12 (ATCC CRL 9595), and the like eucaryotic tissue culture celllines.

Transformation of appropriate cell hosts with a recombinant DNA moleculeof the present invention is accomplished by well known methods thattypically depend on the type of vector used. With regard totransformation of procaryotic host cells, see, for example, Cohen etal., Proc. Natl. Acad. Sci. USA, 69:2110 (1972); and Maniatis et al.,Molecular Cloning, A Laboratory Mammal, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y. (1982).

With regard to transformation of vertebrate cells with vectorscontaining rDNAs, see, for example, Graham et al., Virol., 52:456(1973); Wigler et al., Proc. Natl. Acad. Sci. USA, 76:1373-76 (1979),and the teachings herein.

Successfully transformed cells, i.e., cells that contain a rDNA moleculeof the present invention, can be identified by well known techniques.For example, cells resulting from the introduction of an rDNA of thepresent invention can be cloned to clonally homogeneous cell populationsthat contain the rDNA. Cells from those colonies can be harvested, lysedand their DNA content examined for the presence of the rDNA using amethod such as that described by Southern, J. Mol. Biol., 98:503 (1975)or Berent et al., Biotech., 3:208 (1985).

In addition to directly assaying for the presence of rDNA, successfultransformation can be confirmed by well known immunological methods whenthe rDNA is capable of directing the expression of cortistatin or by thedetection of cortistatin binding activity.

For example, cells successfully transformed with an expression vectorproduce proteins displaying cortistatin antigenicity or biologicalactivity. Samples of cells suspected of being transformed are harvestedand assayed for either cortistatin biological activity or antigenicity.

Thus, in addition to the transformed host cells themselves, the presentinvention also contemplates a culture of those cells, preferably amonoclonal (clonally homogeneous) culture, or a culture derived from amonoclonal culture, in a nutrient medium. Preferably, the culture alsocontains a protein displaying cortistatin antigenicity or biologicallyactivity.

Nutrient media useful for culturing transformed host cells are wellknown in the art and can be obtained from several commercial sources. Inembodiments wherein the host cell is mammalian, a “serum-free” mediumcan be used.

H. Screening Methods to Identify Agonists and Antagonists of Cortistatin

The ability to selectively bind/modulate function of a cortistatinreceptor by a cortistatin ligand is at the heart of useful cortistatinpharmacology, and depends on identifying pharmacological molecules whichcan act a selective ligands, agonists or antagonists for a cortistatinreceptor. To that end, the elucidation of new cortistatin proteins, suchas those described herein, provides valuable tools for the search forselective reagents, tools that are useful in binding assays, and inscreening assays which indicate selective drug response to thecortistatin receptor.

Thus, the invention contemplates methods for determining whether amolecule binds to, and preferably whether the molecule activates, apreselected cortistatin receptor.

The method comprises conducting a binding assay to identify moleculeswhich bind the cortistatin receptor, as described in any of the assaysherein. Thus, the method comprises (1) contacting a candidate moleculewith a cell having a cortistatin receptor under conditions permittingbinding of cortistatin to the receptor, and (2) detecting the presenceof the candidate molecule bound to the cortistatin receptor, therebydetermining whether the candidate binds to the receptor. The receptor istypically a cell surface protein when expressed by the cells.

Alternatively, one can use a competition format to identify analogs ofcortistatin by using a labeled cortistatin, and measuring the amount ofbound label in the presence of a candidate ligand, indicating whetherthe candidate competes with labeled cortistatin for binding to thereceptor. An exemplary competition assay is described herein.

It is also possible to use the above method to determine whether themolecule which binds to the cortistatin receptor also activates ormotivates the receptor's function, i.e., acts as an agonist, ordetermine whether the molecule inhibits the receptor's function, i.e.,acts as an antagonist. Thus, by evaluating in the detecting step whetherthe cortistatin receptor is activated, one determines whether thecandidate molecule is bioactive.

Methods for detecting bioactivity of the candidate molecule can vary,but typically involve measuring changes in intracellular levels of asecondary messenger effected as a result of binding, detecting changesin electrical potential, observing physiological or behavioral effectsrelated to cortistatin function, and the like methods. Exemplary assaysfor binding or for cortistatin-specific bioactivity are described in theExamples and include measurement of hyperpolarization of hippocampalcells, measurement of induction of slow wave sleep waveform two (SWS2),direct binding to a cell having a cortistatin receptor, assayingaccumulation of cAMP, and the like assays.

It is noted that the cortistatin receptor has not been characterized inextensive detail. It is known that cortistatin binds to the somatostatinreceptor, for which there are several species of receptor, and it isknown that cortistatin also binds a more specific receptor, designatedfor convenience as the “true” cortistatin receptor, although the bindingreaction with the somatostatin receptor is likely a real physiologicalevent, making it a “true” receptor for some purposes. Thus, any receptorthat binds cortistatin can be referred to as a cortistatin receptor forthe purposes of a screening assay, although receptors with the highestaffinity and specificity for cortistatin are preferred. In practicingthe present screening methods, one can use any of a variety of cellslines or tissues that possess a cortistatin receptor, including theexemplary cell lines and tissues described herein. The invention shouldnot be construed as limiting so long as the binding or bioactivity assayinvolves the use of a cortistatin receptor. In preferred embodiments, areceptor that is specific for cortistatin should be used. Specificitycan be demonstrated by well known methods of ligand binding andligand-mediated activation.

Thus, a related embodiment contemplates a method for screening toidentify a candidate molecule that can bind, inhibit or activate apreselected cortistatin receptor by functioning as a cortistatin agonistor antagonist. The method comprises:

(a) contacting a mammalian cell with said candidate drug underconditions permitting activation of said cortistatin receptor bycortistatin; and

(b) detecting the activation status of said cortistatin receptor, andthereby determining whether the drug activates or inhibits saidreceptor.

I. Methods for Altering Cortistatin Receptor Function

1. Therapeutic Methods

It is contemplated that the certain reagents described in the presentinvention have the capacity to modulate cortistatin receptor function,such as agonists or antagonists, and therefore are useful in therapeuticmethods for conditions mediated by the cortistatin receptor.

Cortistatin polypeptides that mimic exposed regions of cortistatin havethe ability to function as analogs and compete for binding to thecortistatin receptor, or for other agents that would normally interactwith the receptor, thereby inhibiting binding of cortistatin to thereceptor.

Furthermore, antibodies and monoclonal antibodies of the presentinvention that bind to exposed regions of cortistatin have the capacityto alter cortistatin receptor function by blocking natural interactionswith cortistatin that normally interact at the site. Exemplaryantibodies are the anti-cortistatin antibodies described earlier.

Finally, oligonucleotides are described herein which are complementaryto mRNA that encodes a cortistatin protein of this invention and thatare useful for reducing gene expression and translation of thecortistatin mRNA, thereby altering cortistatin levels in a tissue.

Thus, in one embodiment, the present invention provides a method formodulating cortistatin function in a animal or human patient comprisingadministering to the patient a therapeutically effective amount of aphysiologically tolerable composition containing a cortistatinpolypeptide, analog or peptidomimetic, anti-cortistatin antibody ormonoclonal antibody, cortistatin agonist or antagonist, or anoligonucleotide of the present invention.

A therapeutically effective amount of a cortistatin polypeptide, as anexample for practicing the invention, is a predetermined amountcalculated to achieve the desired effect, i.e., to inhibit receptorinteraction with its normal target, and thereby interfere with normalreceptor function.

Similarly, a therapeutically effective amount of an anti-cortistatinantibody is a predetermined amount calculated to achieve the desiredeffect, i.e., to immunoreact with the cortistatin, and thereby inhibitthe cortistatin receptor's ability to interact with its normal target,cortistatin, and thereby interfere with normal receptor function.

The in vivo inhibition of cortistatin receptor function using acortistatin polypeptide, an anti-cortistatin antibody, or cortistatinagonist or antagonist of this invention is a particularly preferredembodiment and is desirable in a variety of clinical settings, such aswhere the patient is exhibiting symptoms of an over or under activatedcortistatin receptor.

A therapeutically effective amount of a cortistatin polypeptide, agonistor antagonist of this invention is typically an amount such that whenadministered in a physiologically tolerable composition is sufficient toachieve a plasma concentration of from about 0.1 micromolar (μM) toabout 100 μM, and preferably from about 0.5 μM to about 10 μM.

A therapeutically effective amount of an antibody of this invention istypically an amount of antibody such that when administered in aphysiologically tolerable composition is sufficient to achieve a plasmaconcentration of from about 0.1 microgram (μg) per milliliter (ml) toabout 100 μg/ml, preferably from about 1 μg/ml to about 5 μg/ml, andusually about 5 μg/ml.

The effectiveness of the therapy can be determined by observing ablationof the symptoms associated with the function of the cortistatin receptorbeing inhibited.

The therapeutic compositions containing a cortistatin polypeptide,agonist, antagonist or anti-cortistatin antibody of this invention areconventionally administered intravenously or by a method for delivery toa brain tissue, as by injection of a unit dose, for example. The term“unit dose” when used in reference to a therapeutic composition of thepresent invention refers to physically discrete units suitable asunitary dosage for the subject, each unit containing a predeterminedquantity of active material calculated to produce the desiredtherapeutic effect in association with the required diluent; i.e.,carrier, or vehicle.

Delivery to a brain tissue or CSF can be accomplished by a variety ofmeans, including by direct injection, by use of a cannula into thetarget tissue, by direct application in a surgical procedure, byadsorption across the blood-brain barrier following intravenousadministration, and the like means.

The therapeutic compounds and compositions are generally administered soas to contact the cells or the tissue containing cells which contain thetarget cortistatin receptor. This administration can be accomplished byintroduction of the composition internally such as orally,intravenously, intramuscularly, intranasally or via inhalation ofaerosols containing the composition, and the like, by cannula into abrain tissue, or by introduction into or onto a tissue system as byintroduction transdermally, topically or intralesionally, insuppositories, or by intra-orbital injection, and the like.

The compositions are administered in a manner compatible with the dosageformulation, and in a therapeutically effective amount. The quantity tobe administered depends on the subject to be treated, capacity of thesubject's system to utilize the active ingredient, and degree oftherapeutic effect desired. Precise amounts of active ingredientrequired to be administered depend on the judgement of the practitionerand are particular to each individual. However, suitable dosage rangesfor systemic application are disclosed herein and depend on the route ofadministration. Suitable regimes for initial administration and boostershots are also variable, but are typified by an initial administrationfollowed by repeated doses at one or more hour intervals by a subsequentinjection or other administration. Alternatively, continuous intravenousinfusion sufficient to maintain concentrations in the CSF or blood inthe ranges specified for in vivo therapies are contemplated.

As an aid to the administration of effective therapeutic amounts of acortistatin polypeptide, agonist, antagonist, antibody, or monoclonalantibody, (hereinafter a “therapeutic agent”) a diagnostic method ofthis invention for detecting a therapeutic agent in the subject's CSF orblood is useful to characterize the fate of the administered therapeuticagent. Suitable diagnostic (monitoring) assays are described herein.

2. Methods for Inhibiting Gene Expression

In another embodiment, the invention contemplates the use of nucleicacids encoding portions of a cortistatin gene for inhibiting geneexpression and function.

Thus, the present invention provides for a method for inhibitingexpression of cortistatin gene products and thereby inhibiting thefunction of the target cortistatin protein. The DNA segments and theircompositions have a number of uses, and may be used in vitro or in vivo.In vitro, the compositions may be used to block function and/orexpression of cortistatin in cell cultures, tissues, organs and the likematerials that can express cortistatin. In vivo, the compositions may beused prophylactically or therapeutically for inhibiting expression of acortistatin gene, and by inhibiting diseases or medical conditionsassociated with the expression or function of the cortistatin gene orthe activity state of its receptor.

The method comprises, in one embodiment, contacting cells or tissueswith a therapeutically effective amount of a pharmaceutically acceptablecomposition comprising a DNA segment of this invention. In a relatedembodiment, the contacting involves introducing the DNA segmentcomposition into cells expressing a cortistatin protein.

The DNA segment can be in a variety of forms, but is preferably in asingle-stranded form to facilitate complementary hybridization to thetarget mRNA in the cell in which the cortistatin gene expression is tobe altered.

The term “cells” is intended to include a plurality of cells as well assingle cells. The cells can be isolated, or can be cells that form alarger organization of cells to form a tissue or organ.

In a further embodiment, the invention contemplated the method ofinhibiting the expression of cortistatin genes in a patient comprisingadministration to the patient of a therapeutically effective amount of aDNA segment composition of this invention in a pharmaceuticallyacceptable excipient. In cases where the distribution of the cortistatinis believed to be disseminated in the body, the administration oftherapeutic oligonucleotide can be systemic. Alternatively, the targetcortistatin can be localized to a tissue, and the therapeutic method canlikewise be directed at delivering the therapeutic DNA segment to thetissue to be treated.

The concentration of the active DNA segment ingredient in a therapeuticcomposition will vary, depending upon the desired dosage, use, frequencyof administration, and the like. The amount used will be atherapeutically effective amount and will depend upon a number offactors, including the route of administration, the formulation of thecomposition, the number and frequency of treatments and the activity ofthe formulation employed.

The use of therapeutic DNA segments, and therefore the delivery of thoseDNA segments into cells where they are effective, has been described ina variety of settings. It is generally known that therapeuticallyeffective intracellular levels of nucleic acids, and particularlysmaller nucleic acids such as DNA segments and oligonucleotides, can beachieved by either exposing cells to solutions containing nucleic acidsor by introduction of the nucleic acids into the inside of the cell.Upon exposure, nucleic acids are taken up by the cell where they exerttheir effectiveness. In addition, direct introduction into the cell canbe provided by a variety of means, including microinjection, delivery bythe use of specific uptake vehicles, and the like.

The pharmaceutical composition containing the therapeuticoligonucleotide preferably also contains physiologically acceptablecarriers, in particular hydrophobic carriers which facilitate carryingthe oligonucleotide through the cell membrane or blood brain barrier.

Exemplary descriptions of the delivery of therapeutic DNA segments andoligonucleotides into cells can be found in the teachings of U.S. Pat.Nos . 5,04,820, 4,806,463, 4,757,055, and 4,689,320, which teachings arehereby incorporated by reference.

A therapeutically effective amount is a predetermined amount calculatedto achieve the desired effect, i.e., to bind to a cortistatin genepresent and thereby inhibit function of the gene.

As is apparent to one skilled in the art, the copy number of acortistatin gene may vary, thereby presenting a variable amount oftarget with which to hybridize. Thus it is preferred that thetherapeutic method achieve an intracellular concentration of atherapeutic DNA segment of this invention in molar excess to the copynumber of the gene in the cell, and preferably at least a ten-fold, morepreferably at least a one-hundred fold, and still more preferably atleast a one thousand-fold excess of therapeutic DNA segments relative tothe gene copy number per cell. A preferred effective amount is anintracellular concentration of from about 1 nanomolar (nM) to about 100micromolar (μM), particularly about 50 nM to about 1 μM.

Alternatively, a therapeutically effective amount can be expressed as anextracellular concentration. Thus it is preferred to expose an cellcontaining a cortistatin gene to a concentration of from about 100 nM toabout 10 millimolar (mM), and preferably about 10 μM to 1 mM. Thus, inembodiments where delivery of a therapeutic DNA segment composition isdesigned to expose cells to the nucleic acid for cellular uptake, it ispreferred that the local concentration of the DNA segment in the area ofthe tissue to be treated reach the extracellular concentrations recitedabove.

For patient dosages, using a 20 nucleotide base double-stranded DNAsegment as the standard, a typical dosage of therapeutic composition fora 70 kilogram (kg) human contains in the range of about 0.1 milligram(mg) to about 1 gram of 20-mer DNA segment per day, and more usually inthe range of about 1 mg to 100 mg per day. Stated differently, a dosageof about 1 μg/kg/day to about 15 mg/kg/day, and preferably about 15 to1500 μg/kg/day is contemplated.

The in vivo inhibition of cortistatin gene expression and/or function bya therapeutic composition of this invention is desirable in a variety ofclinical settings, such as where the patient is at risk for diseasebased on expression of the cortistatin gene.

3. Therapeutic Compositions

The present invention contemplates therapeutic compositions useful forpracticing the therapeutic methods described herein. Therapeuticcompositions of the present invention contain a physiologicallytolerable carrier together with a therapeutic reagent of this invention,namely a cortistatin polypeptide, an anti-cortistatin antibody ormonoclonal antibody, or oligonucleotide as described herein, dissolvedor dispersed therein as an active ingredient. In a preferred embodiment,the therapeutic composition is not immunogenic when administered to amammal or human patient for therapeutic purposes.

As used herein, the terms “pharmaceutically acceptable”,“physiologically tolerable” and grammatical variations thereof, as theyrefer to compositions, carriers, diluents and reagents, are usedinterchangeably and represent that the materials are capable ofadministration to or upon a mammal without the production of undesirablephysiological effects such as nausea, dizziness, gastric upset and thelike.

The preparation of a pharmacological composition that contains activeingredients dissolved or dispersed therein is well understood in theart. Typically such compositions are prepared as injectables either asliquid solutions or suspensions, however, solid forms suitable forsolution, or suspensions, in liquid prior to use can also be prepared.The preparation can also be emulsified.

The active ingredient can be mixed with excipients which arepharmaceutically acceptable and compatible with the active ingredientand in amounts suitable for use in the therapeutic methods describedherein. Suitable excipients are, for example, water, saline, dextrose,glycerol, ethanol or the like and combinations thereof. In addition, ifdesired, the composition can contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents, pH buffering agentsand the like which enhance the effectiveness of the active ingredient.

The therapeutic composition of the present invention can includepharmaceutically acceptable salts of the components therein.Pharmaceutically acceptable salts include the acid addition salts(formed with the free amino groups of the polypeptide) that are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, tartaric, mandelic and the like.Salts formed with the free carboxyl groups can also be derived frominorganic bases such as, for example, sodium, potassium, ammonium,calcium or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.

Physiologically tolerable carriers are well known in the art. Exemplaryof liquid carriers are sterile aqueous solutions that contain nomaterials in addition to the active ingredients and water, or contain abuffer such as sodium phosphate at physiological pH value, physiologicalsaline or both, such as phosphate-buffered saline. Still further,aqueous carriers can contain more than one buffer salt, as well as saltssuch as sodium and potassium chlorides, dextrose, polyethylene glycoland other solutes.

As described herein, for intracellular delivery of oligonucleotides,specialized carriers may be used which facilitate transport of theoligonucleotide across the cell membrane. These typically arehydrophobic compositions, or include additional reagents which targetdelivery to and/or into cells.

Liquid compositions can also contain liquid phases in addition to and tothe exclusion of water. Exemplary of such additional liquid phases areglycerin, vegetable oils such as cottonseed oil, and water-oilemulsions.

A therapeutic composition contains an amount of a cortistatinpolypeptide or anti-cortistatin antibody molecule of the presentinvention sufficient to inhibit cortistatin function. Typically this isan amount of at least 0.1 weight percent, and more preferably is atleast 1 weight percent, of peptide or antibody per weight of totaltherapeutic composition. A weight percent is a ratio by weight ofpeptide or antibody to total composition. Thus, for example, 0.1 weightpercent is 0.1 grams of polypeptide per 100 grams of total composition.

EXAMPLES

The following examples are intended to illustrate but are not to beconstrued as limiting the specification and claims in any way.

1. Identification of Cortistatin Nucleic Acids

A. Rat cDNA

To screen for novel mRNAs, an 140 base pair (bp) cDNA clone was obtainedfrom a subtracted rat hippocampal library and then used as a probe toscreen a rat brain cDNA library in the plasmid pHG327 as described byForss-Petter et al., J. Mol. Neurosci., 1:63-75 (1989). The cDNA librarywas constructed as described by Staeheli et al., Cell, 44:147-158(1986), the disclosure of which is hereby incorporated by reference.

Briefly, the subtracted cDNA library was constructed essentially asdescribed by Usui et al., J. Neurosci., 14:4915-4926 (1994) and was theresult from subtracting a cDNA library made from hippocampi of rats thathad been stimulated at high frequency in vivo (referred to as the targetlibrary in Usui et al.) with a cDNA library made from the contralateralhippocampi of the same rats (referred to as the driver cDNA library).Clones from this subtracted library were arrayed on nylon replicafilters and hybridized with probes consisting of the target and drivercDNA libraries. cDNA clones hybridizing with the target but not thedriver were further analyzed by dideoxy sequencing (Sanger et al. Proc.Natl. Acad. Sci. USA, 74:5463-5467 (1977) and in situ hybridization (deLecea et al., Mol. Brain Res., 25:286-296 (1994). The 140 base pair (bp)long nucleotide sequence of clone 1D4 (later named preprocortistatin),that was used as the above-described screening probe, was then comparedwith sequences in the GenBank database and was recognized by having asignificant degree (82%) of similarity with the nucleotide sequencereported for somatostatin.

Further screens of whole rat brain and hippocampal cDNA librariesproduced five additional clones up to 438 nucleotides in length,including two displaying an initiator methionine codon. The nucleotidesequence of the isolated clones was determined using the dideoxy methodas described by Sanger et al., Proc. Natl. Acad. Sci., USA, 74:5463-5467(1977). Sequence alignment was performed with the BESTFIT program (GCGgroup, University of Wisconsin).

From the five cDNA clones obtained from screening the above-identifiedlibraries, a complete coding nucleotide sequence of ratpreprocortistatin cDNA, 438 nucleotides in length, was compiled as shownin FIG. 1 and listed in SEQ ID NO 1. The complete preprocortistatin cDNAclone displays a 336 nucleotide open reading frame (ORF) with aN-terminal signal peptide whose cleavage site is indicated by an arrowbetween amino acid positions 27 and 28 corresponding to a cleavage siteafter nucleotide position 110. A sequence of six iterations of thetrinucleotide CTG repeats encoding leucine residues contained within thecoding region for the signal peptide is underlined. Such a tripletexpansion in other genes has been implicated as causal in neurologicaldiseases, e.g., myotonic dystrophy as described by Brook et al., Cell,68:799-808 (1992).

Translation of this rat cDNA sequence indicated that a novel protein of112 amino acid residues, called rat preprocortistatin, was encoded asshown in FIG. 1 aligned under the cDNA sequence. The deduced amino acidsequence of rat preprocortistatin is also listed in SEQ ID NO 1 with thenucleotide sequence and in SEQ ID NO 2 alone.

Cleavage of the preprospecies to procortistatin would produces a matureprotein that is processed at either of two tandem basic amino acidpairs, KK (lys-lys) or KR (lys-arg) shown in bold in FIG. 1, to producecortistatin-29 and cortistatin-14, the latter shown in FIG. 1 in thesolid lined box, analogous to the cleavage of preprosomatostatin at 28and 14 residues as described by Glushankov et al., Proc. Natl. Acad.Sci., USA, 81:6662-6666 (1984), or at both basic pairs to additionallyproduce cortistatin-13, shown in FIG. 1 with the hatched lined box.Whereas cortistatin-13 is unrelated to known species, cortistatin-14shares 11 of 14 residues with somatostatin-14. Alignment of ratpreprocortistatin with the 116 amino acid residue sequence ofpreprosomatostatin is shown in FIG. 2 labeled respectively as CST andSST. Single dots between the sequences indicate non-conservative aminoacid substitutions while double vertical dots indicate conservativeamino acid substitutions. Vertical lines between the sequences indicateidentity between CST and SST. Single dots between the continuoussequence of CST indicate spacing to accommodate for the alignment withSST. The amino acid residue sequence of CST shown in FIG. 2 correspondsto positions 10 to 112 in SEQ ID NO 2. The amino acid residue sequenceof SST is listed in SEQ ID NO 3.

The shared amino acids between preprocortistatin and preprosomatostatininclude two cysteine residues that are likely to render the peptidecyclic and the FWKT amino acid residues (SEQ ID NO 2 from position 104to 107) that are critical for somatostatin binding to its receptors asdescribed by Veber et al., Nature, 280:512-514 (1979). However, extendedamino acid identity is seen only at their C-termini. Both statins sharethe critical residues for binding to the receptors as shown in bold inthe figure and the cysteines that are likely to render the peptidescyclic.

As shown in FIG. 2, for the portion of the preprostatin sequencesbeginning after the bold KK for CST and the bold RK for SST indicatingpeptides with 14 amino acids, the cortistatin-14 and somatostatin-14 arepermuted by one amino acid. Thus, the alignment of cortistatin begins atthe second amino acid of the somatostatin-14 peptide, corresponding toamino acid residue position 104 in FIG. 2, and cortistatin terminateswith a lysine residue that extends one amino acid residue, correspondingto CST amino acid position 112, beyond the C-terminal cysteine ofsomatostatin. This difference and their cDNA sequences indicate clearlythat they are the products of separate genes.

B. Mouse cDNA

A mouse (C57/B16) cerebral cortex cDNA library, constructed in thepT7T3D vector (Pharmacia, Piscataway, N.J.) was similarly screened toobtain the mouse homolog to the rat preprocortistatin cDNA obtainedabove. The screens produced four additional clones up to 430 nucleotidesin length, including two displaying a initiator methionine codon. Thenucleotide sequence of the isolated clones was determined as describedabove as was alignment of the sequences.

From the four cDNA clones obtained from screening the above-identifiedlibraries, a complete coding nucleotide sequence of mousepreprocortistatin cDNA, 427 nucleotides in length, was compiled as shownin FIG. 3 and listed in SEQ ID NO 4. The complete preprocortistatin cDNAclone displays a 327 nucleotide open reading frame (ORF) with aN-terminal signal peptide whose cleavage site is indicated by an arrowbetween amino acid positions 25 and 26 corresponding to a cleavage siteafter nucleotide position 99. A sequence of three iterations of thetrinucleotide CTG encoding leucine residues contained within the codingregion for the signal peptide is underlined.

Translation of this mouse cDNA sequence indicated that a novel proteinof 109 amino acid residues, called mouse preprocortistatin, was encodedas shown in FIG. 3 aligned under the cDNA sequence. The deduced aminoacid sequence of mouse preprocortistatin is also listed in SEQ ID NO 4with the nucleotide sequence and in SEQ ID NO 5 alone.

Similar to the rat preprocortistatin, cleavage of the mousepreprospecies to procortistatin produces a mature protein that isprocessed at either of two tandem basic amino acid pairs, KS (lys-ser)and KK (lys-lys) shown in bold in FIG. 3, to produce mousecortistatin-29 and mouse cortistatin-14, the latter shown in FIG. 3 inthe solid lined box, or at both basic pairs to additionally producemouse cortistatin-13, shown in FIG. 3 with the hatched lined box.

After introduction of two gaps, the mouse and rat nucleotide sequenceswere 86% identical. Assuming that the mouse translation initiationproduct begins at the second methionine triplet, it contains 108 aminoacids compared to 112 for rat. Again, after introduction of two gaps,the encoded rat and mouse proteins share 82% identity. The mousenucleotide sequence corresponding to cortistatin-14 and the adjacentlysine doublet that serves as its site of proteolytic release from itsprecursor were identical to same region in the rat sequence, thussupporting a functional conservation of the mature peptide. The DNAsequence upstream from the processing site of cortistatin-14 showedseveral points of divergence, including some resulting innon-conservative amino acid substitutions. The sequence corresponding tothe signal peptide of preprocortistatin contains only three iterationsof CTG encoding the amino acid leucine, in contrast to six iterations ofthe same triplet in the rat peptide precursor, indicating that thissequence is unstable and subject to expansion.

C. Human cDNA

The human homolog is similarly obtained from screening human brain cDNAlibraries essentially as described above for rat and mouse cortistatinnucleic acids and proteins. To screen for novel mRNAs, a 120 bp fragmentof the human coding sequence was isolated by PCR using degenerateprimers from the mouse and rat sequences. The nucleotide sequence ofthis fragment was compared to the EST database and one sequence wasfound with significant similarity to cortistatin. A 250 bp nucleotidefragment was obtained and used as a probe to screen a human whole braincDNA library. The screens produced two cDNA clones, 450 and 270nucleotides in length, and the sequence from the longest was determined.Human preprocortistatin cDNA was amplified by PCR using primers to theC-terminal sequence of cortistatin. The PCR fragment was cloned, randomprime labeled and used to screen a cDNA library prepared from humanwhole brain mRNA (Clontech).

A complete coding nucleotide sequence of human preprocortistatin cDNA,701 nucleotides in length, was compiled as shown in FIG. 3a and listedin SEQ ID NO 25. A sequence of four iterations of the trinucleotide CTGencoding leucine residues contained within the coding region for thesignal peptide is underlined in FIG. 3a. The complete preprocortistatincDNA clone displays a 315 ORF, which begins at position 78 of SEQ ID NO25.

Translation of this human cDNA sequence indicated that a novel proteinof 105 amino acid residues, called human preprocortistatin, was encodedas shown in FIG. 3b. The deduced amino acid sequence of humanpreprocortistatin is listed in SEQ ID NO 26.

Similar to the rat preprocortistatin, cleavage of the humanpreprospecies to procortistatin generates a mature protein that isprocessed at either of two RR (arg-arg) tandem basic amino acid pairs,to produce human cortistatin-29 and human cortistatin-17. The humanpreprospecies along with the prospecies and the mature proteins arelisted in Table 1 in the Examples including their noted amino acidresidue sequences.

The human and rat nucleotide sequences are 71% identical. The humancortistatin-17 shares 13 of the last 14 residues with rat and mousecortistatin-14. The lysine doublet that lies just N-terminal tocortistatin-14 in the rat and mouse is not conserved in the humansequence. The DNA sequence upstream from the processing site ofcortistatin-14 are not very conserved across species. However, ratcortistatin-31 and human cortistatin-31 share 13 residues clustered intheir N-terminal regions that are conserved among the rat, mouse, andhuman prohormone sequences. The sequence corresponding to the signalpeptide of preprocortistatin contains only four iterations of CTGencoding the amino acid leucine, in contrast to six iterations of thesame triplet in the rat peptide precursor or three in mouse, indicatingthat this sequence is unstable and subject to expansion.

2. Preparation of Cortistatin Protein and Polypeptides

A. Recombinant Proteins

Rat preprocortistatin (SEQ ID NO 1) is inserted into the BamH1 sites ofthe pHG237 vector, both the DNA and vector are described above. Upondigestion with BamH1 restriction enzyme, the resultant 450 bp fragmentis then inserted directly into the BglII site of the polylinker regionof the pCM4 vector (David W. Russell, Dept. of Molecular Genetics,University of Texas Southwestern Medical Center, Dallas, Tex.). Thisvector uses the cytomegalovirus (CMV) promoter to direct the expressionof foreign proteins in mammalian cells. Several eight to ten amino acidepitope “tags” are added by PCR to the N-terminal of preprocortistatinto allow visualization of the processed product in mammalian cells.

For example, the respective 5′ and 3′ synthetic oligonucleotides,written in the 5′ to 3′ direction, 5′ ATCGAGATCTAAGGAGGATGGGTGGCTGCAG3′(SEQ ID NO 13) and 5′ACTGTCTAGATCATAGGTCTTCTTCTGATATTAGTTTTTGTTCCTTGCACGA GGAGAAGGTTTTCCAG3′(SEQ ID NO 14) are used as primers in PCR to amplify preprocortistatinbeginning at nucleotide position 23 in SEQ ID NO 1 with an insertedBglII site added at its 5′ end to the 3′ end having an inserted c-mycepitope tag. The 5′ primer is also referred to as an upstream, sense orforward primer. The 3′ primer is also referred to as a downstream,anti-sense or backward primer. The resultant PCR products are such that,when subcloned into the pCMV or related vectors and transfected intomammalian cells (CHO, AtT-20 or GH4 cells), produce a procortistatin-myctagged protein product that is visualized by Western blot orimmunocytochemistry, without the need of cortistatin-specificantibodies. For in vivo visualization of the processing, thepreprocortistatin sequence amplified as described above could beinserted into the pGFP-N1 vector (Clontech, Palo Alto, Calif.), whichcontains the green fluorescein protein (GFP) from Aequorea victoria.

Procortistatin proteins for use in this invention are also produced inbacteria and purified by subcloning the procortistatin coding sequencedescribed above and seen in FIG. 1 into the pRSET B vector (Invitrogen,San Diego, Calif.), which contains the nucleotide sequence encoding 6histidines before the insertion of the procortistatin sequence. Thevector contains the T7 promoter which drives the expression of6xHis-tagged proteins in E. coli. For this aspect, the respectivesynthetic 5′ and 3′ oligonucleotides, 5′ ATCGAGATCTGTCCTGGAGA3′ (SEQ IDNO 15) and 5′ ACTGAATTCAGGCCACGGCTGCATTCACAG 3′ (SEQ ID NO 16), are usedas primers in PCR to amplify the rat preprocortistatin sequence withoutthe signal peptide into the BglII and EcoRI sites of the PRSET B vector.Once expressed, the expressed 6× histidine-tagged procortistatinsequence is then purified by affinity chromatography on a TALON(Clontech) metal affinity resin.

A procortistatin-glutathione-S-transferase fusion protein (CST-GST) isproduced in E. coli by subcloning the procortistatin sequence into theappropriate sites of the pGEX2 vector (Pharmacia), as described above.

Thus, the methods described herein are useful for the generation of bothrecombinant cortistatin proteins and recombinant cortistatin fusionproteins. With the above-described expression methods, mouse and humanhomologs of the rat procortistatin are similarly prepared along with theremaining rat and mouse cortistatin proteins and peptides listed belowin Table 1. The cloning and expression of the cortistatin proteins andpolypeptides of this invention are techniques well known to one ofordinary skill in the art and are described, for example, in “CurrentProtocols in Molecular Biology”, eds. Ausebel et al., Wiley & Sons,Inc., New York (1989), the disclosures of which are hereby incorporatedby reference.

Once expressed, the recombinant cortistatin proteins and polypeptidesalong with fusion proteins thereof are useful in the screening methods,diagnostic methods and therapeutic modalities as described below.Specifically, with respect to screening methods, recombinantcortistatins are used in the solid phase in assays including Westernblot, ELISA, RIA, and the like, all of which are well known techniquesin the art. Similarly, the molecules described herein are used in liquidphase in assays including receptor binding assays for direct binding orfor competition of binding (see Example 5), for cAMP activation assays(see Example 5), for identifying cortistatin-specific receptors (seeExample 7), and the like. The determination and identification ofcortistatin analogs and antagonists is also facilitated with the use ofrecombinant cortistatin proteins and polypeptides as described inExample 6. Therapeutic uses of recombinant molecules are similarlydescribed in Example 8. Other uses not described herein of the moleculesof the present invention are also contemplated.

B. Synthetic Proteins and Polypeptides

An alternative method to preparing recombinant cortistatin proteins andpolypeptide is preparing synthetic versions thereof. For this procedure,the polypeptides were synthesized using standard solid-phase synthesistechniques as, for example, described by Merrifield, Adv. Enzymol.,32:221-296 (1969), and Fields, G. B. and Noble, R. L., Int. J. PeptideProtein Res., 35:161-214 (1990) and as described in U.S. Pat. Nos.4,900,811 and 5,242,798, the disclosures of which are herebyincorporated by reference.

The various cortistatin proteins and peptides of this invention arehereinafter referred to by their designations as listed in Table 1. Thecorresponding SEQ ID NO for each peptide is also listed in Table 1.

TABLE 1 SEQ ID Designation NO FIG. rat preprocortistatin 2 1-completesequence rat procortistatin 6 1-from arrow to end rat cortistatin-29 71-from beginning of hatched line to end rat cortistatin-14 8 1-in solidbox rat cortistatin-13 9 1-in hatched box mouse preprocortistatin 53-complete sequence mouse procortistatin 10 1-from arrow to end mousecortistatin-29 11 3-from beginning of hatched line to end mousecortistatin-14 8 3-in solid box mouse cortistatin-13 12 3-in hatched boxhuman preprocortistatin 26 3a-complete sequence human cortistatin-29 263a-from arrow to end human cortistatin-17 26 3a-in solid box humancortistatin-31 26 3a-in hatched box

The cortistatin-14 peptide, having the same amino acid residue sequenceforrat and mouse, was synthesized in the carboxy-terminal amide form. Itwas then analyzed by reverse phase high performance liquidchromatography (HPLC) on a Vydac C-18 column (Alltech Associates, Inc.,IL) with a 0-60% acetonitrile linear gradient in 0.1% trifluoroaceticacid. The peptide was then purified to homogeneity by preparative HPCLusing optimal conditions suggested by the analytical chromatography. Theamino acid composition and concentration of the isolated peptide wasdetermined with a 24 hour hydrolysis in 6 N HCl in evacuated tubes at110 degrees Celsius (110° C.) and subsequent analysis on a Beckman Model6300 High Performance Analyzer.

After purification, the peptide was separately resuspended in distilledwater to form a dissolved peptide solution at a final concentration of2.5 mM. Subsequently, one-tenth volume of 10-fold concentrated buffer,referred to as TBS-Az containing 0.05 M Tris hydroxymethylaminomethane-hydrochloride (Tris-HCl) at pH 7.4 0.1 M sodium chloride(NaCl), 0.02% sodium azide (NaN₃), was added. The pH of the solution waschecked, and if necessary, adjusted to pH 7.4 with titrated amounts of 1M Tris-base.

The remaining cortistatin proteins and peptides listed in Table 1 aresimilarly synthesized and purified for use in practicing this invention.

3. Preparation of Anti-Cortistatin Antibodies

A. Preparation of Polyclonal Antisera to Synthetic Polypeptides

1) Preparation of Immunogen

For preparation of a peptide immunogen, the synthetic polypeptidecortistatin-14 was prepared as described in Example 2. The synthesizedpeptide was coupled to edestin (Sigma, St. Louis, Mo.) using theheterobifunctional crosslinking agent,N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) (PierceBiochemicals, Rockford, Ill.). For the coupling procedure, 80microliters (μl) of 10 milligrams/milliliter (mg/ml) SPDP dissolved indimethylformamide was admixed dropwise to 400 μl 15 mg/ml edestin in 0.1M phosphate, 0.1 M NaCl at pH 8.5 under continuous stirring conditionsfor 30 minutes at 22° C. in order to form SPDP-activated edestin.

The resultant SPDP-activated edestin was then extensively dialyzed at 4°C. against a buffered solution of 0.1 M phosphate and 0.1 M NaCl at pH7.4 in order to remove uncoupled SPDP. Six mg of prepared peptide wasfirst dissolved in 2 ml of 0.1 M phosphate and 0.1 M NaCl at pH 7.4 andthen admixed with SPDP-activated edestin prepared above under continuousstirring conditions. The degree of coupling of the peptide with edestinwas monitored by diluting an aliquot of the mix 1:100 at time zero, andeach hour thereafter, and measuring the release of pyridine-2-thione at343 nm in a spectrophotometer. The end point of coupling was determinedto be an increase of 0.2 in absorbency, or upon visualization ofprecipitate at which point edestin conjugates peptide was formed, anddesignated cortistatin-14-edestin immunogen.

To prepare antisera specific for the remaining peptides listed in Table1 and for species-specific preprocortistatin and procortistatin, theprotein and peptide preparations described in Example 2 are similarlyprepared as immunogens.

2) Immunization and Collection of Polyclonal Antisera

To form anti-peptide antibodies, the cortistatin-14-edestin immunogenprepared above was emulsified using Adjuvant Complete Freund (DIFCOLaboratories, Detroit, Mich.) for the first injection and AdjuvantIncomplete Freund (DIFCO) for all subsequent injections according to themanufacturer's instructions, and the immunogen was incorporated into theemulsion at a concentration of 2 mg/ml. One-half ml of a preparedemulsion was injected subcutaneously into each of two New Zealand whiterabbits after pre-immune serum samples are collected. The rabbits wereinjected three times at weekly intervals following the injectionprotocol as detailed. Two weeks after the last injection, blood sampleswere collected to check antibody titer against the specific peptidecortistatin-14-edestin used as an immunogen by the ELISA assay describedbelow in Example 3C. The collected blood samples were stored at 4° C.for 12 hours, after which the samples were centrifuged at 3000×g for 20minutes. The resultant supernatant containing anti-peptide antibodieswas collected, designated polyclonal anti-cortistatin peptide antibodiesand stored at −20° C.

Immunization of separate rabbits for the production of antisera againsteach of the peptides and proteins prepared in Example 2 is performed asdescribed herein. The resultant antisera are then screened by ELISA asdescribed below.

C. ELISA to Screen Antisera Immunoreactivity

The peptide antibody titers and immunospecificity in sera collected fromrabbits in Example 3B is determined in binding assays to nativecortistatin molecules in tissue preparation (i.e., in situ) as describedin the Specification or by an enzyme-linked- immunosorbent-assay (ELISA)as described below. The antigens used in the ELISA include theimmunizing peptide as well as recombinant proteins and polypeptides asdescribed in Example 2.

To determine the immunospecificity of the rabbit antisera obtained inExample 2C, ELISA assays are performed. Briefly, 50 μl of 50 μMconcentrations of cortistatin peptides prepared in Example 1 and listedin Table 1 in a buffer consisting of 0.05 M sodium carbonate (Na₂CO₃)and 0.02% NaN₃ at pH 9.0 are separately admixed into the wells ofmicrotiter plates. The plates are maintained at 37° C. for one hour topermit the antigens to become operatively affixed to the well walls.After washing the antigen-coated wells with TBS, the wells are blockedwith 250 μl well of 10% bovine serum albumin (BSA) (Sigma) in TBS forone hour at 22° C. The blocking solution is then removed and the wellsare subsequently washed five times with 250 μl/well of maintenancebuffer (0.05 M Tris-HCl, 0.1 M NaCl, 0.02% NaN₃, 1 mg/ml BSA, 5 mMCaCl₂, 0.01% Tween 20 at pH 7.4).

Fifty μl of rabbit nonimmune or specific antiserum serially diluted inmaintenance buffer are then admixed to the washed wells to form animmunoreaction admixture, that is maintained for one hour at 37° C. to.allow formation of a solid-liquid phase immunoreaction products. Thewells are then washed three times with maintenance buffer followed byadmixture of 50 μl of 1.0 μg/ml of secondary antibody (polyclonalbiotinylated goat-anti-rabbit-IgG) (Pierce Biohemicals, Rockford, Ill.)diluted in maintenance buffer to each well for the detection ofimmunoreactant products. The plates are maintained for one hour at 37°C. after which time the secondary antibody solution is removed.

After washing the wells as described above, 50 μl of 1.0 μg/mlstreptavidin-alkaline-phosphatase (Pierce Biochemicals) in maintenancebuffer are admixed into each well and maintained for 30 minutes at 37°C. Detection of specific immunoreaction products is obtained byadmixture of 150 μl/well of 5 mg/ml p-nitrophenylphosphate (PNPP)(Pierce Biochemicals) in 0.1 M diethanolamine and 0.02% NaN₃ at pH 9.0followed by measurement of the change in absorbance at 405 nm over timeusing the EL312 Microplate Bio-Kinetics Reader and the KinetiCalcSoftware Program (Biotek Instruments, Inc., VT). Nonspecific binding isconsidered as the measured absorbance in 10% BSA blocked wells whichserve as negative controls without the preceding coating of a specificprotein or peptide. Under the described conditions, nonspecific bindingnever exceeds more than 5% of the specific binding. Rabbit anti-peptideantisera which exhibit immunoreactivity that produced an optical densitychange at 405 nm using the kinetic program as compared to theimmunoreactivity of pre-immune serum toward peptides, is selected foruse as an anti-peptide antibody, and also selected for furtherpurification as described in herein.

Rabbit antisera for the other peptide immunogens are similarly screenedfor immunoreactivity to the respective peptide immunogens. Rabbitantisera which exhibit significant immunoreactivity as compared to thepre-immune sera toward each of the peptide immunogens are furtherpurified and analyzed as described below.

D. Purification of Anti-Cortistatin Peptide Antibodies

Purification of the IgG fraction from rabbit antiserum, which showssignificant reactivity towards the immunizing peptides described aboveis conducted by ammonium-sulfate precipitation (0-45%), followed bypurification of IgG on an ion-exchange Mono Q column (Pharmacia LKB,Piscataway, N.J.) connected to a fast protein liquid chromatography(FPLC) system (Pharmacia). For each antiserum, immunoaffinitypurification of the pooled immunoreactive IgG-fraction is performed bypassing approximately 100 mg of the IgG over a 5 ml column containing 3mg of species-specific cortistatin coupled to Sepharose 4B (Pharmacia).After a thorough washing of the column with five column volumes of 0.05M Tris-HCl and 1 M NaCl at pH 7.4 to remove unbound antibodies, thebound IgG is eluted with two column volumes of 0.1 M glycine-HCl at pH2.5. The eluted protein is monitored by absorbance at 280 nm and the IgGconcentrations determined from the extinction coefficient of 13.5. Theeluted IgG is immediately dialyzed against TBS-Az, concentrated against50% sucrose for approximately three to four hours and once moreextensively dialyzed against TBS-Az to a final concentration of 3-4mg/ml.

E. Preparation of Anti-Cortistatin Monoclonal Antibodies

To prepare monoclonal antibodies to the cortistatin proteins andpolypeptides prepared in Example 2, immunogens for each are prepared asdescribed above. Balb/c ByJ mice (The Scripps Research Institute, LaJolla, Calif.) are immunized intraperitoneally (i.p.) with 50 μg ofprepared immunogen in complete Freund's adjuvant (CFA) followed by asecond and third immunization using the same immunogen, each about threeweeks apart, in incomplete Freund's adjuvant (IFA). The mice receive aboost of 50 μg of the prepared peptide intravenously (i.v.) in normalsaline four days prior to fusion and a second similar perfusion boostone day later.

The animals so treated are sacrificed and the spleen of each mouse isharvested. A spleen cell suspension is then prepared. Spleen cells arethen extracted from the spleen cell suspension by centrifugation forabout ten minutes at 1000 rpm, at 23° C. Following removal of theresultant supernatant, the cell pellet is resuspended in 5 ml coldammonium chloride (NH₄Cl) lysing buffer, and is maintained for about 10minutes.

Ten ml of Dulbecco's Modified Eagle Medium (DMEM) (GIBCO) and HEPES[4-(2-hydroxyethyl)-1-piperidineethanesulfonic acid] buffer are admixedto the lysed cell suspension to form an admixture, and that admixture iscentrifuged for about ten minutes at 1000 rpm at 23° C.

After the resultant supernatant is decanted, the pellet is resuspendedin 15 ml of DMEM and HEPES and is centrifuged for about ten minutes at1000 rpm at 23° C. The above procedure is repeated.

The pellet is then resuspended in 5 ml DMEM and HEPES. An aliquot of thespleen cell suspension is then removed for counting. Fusions wereaccomplished in the following manner using the non-secreting mousemyeloma cell line P3X63Ag 8.653.1, a subclone of line P3x63Ag 8.653(ATCC 1580). With a myeloma to spleen cell ratio of about 1 to 10 orabout 1 to 5, a sufficient quantity of myeloma cells are centrifugedinto a pellet, washed twice in 15 ml DMEM and HEPES, and thencentrifuged for 10 minutes at 1000 rpm at 23° C.

Spleen cells and myeloma cells are combined in round bottom 15 ml tubes.The cell mixture is centrifuged for ten minutes at 1000 rpm at 23° C.and the supernatant is removed by aspiration. Thereafter, 200 μl of 50percent (weight per volume) aqueous polyethylene glycol 4000 molecularweight (PEG); (ATCC Baltimore, Md.) at about 37° C. are admixed with thepellet using a 1 ml pipette with vigorous stirring to disrupt thepellet. The cells are then gently mixed for between 15 and 30 seconds.The resultant cell mixture is centrifuged four minutes at 700 rpm.

At about eight minutes from the time of adding the PEG, 5 ml of DMEMplus HEPES buffer are admixed slowly to the pellet, without disturbingthe cells. After one minute, the resulting admixture is broken up with a1 ml pipette and is maintained for an additional four minutes. Thisadmixture is centrifuged for 7 minutes at 1000 rpm. The resultantsupernatant is decanted, 5 ml of HT (hypoxanthine/thymidine) medium areslowly admixed to the pellet, and the admixture is maintainedundisturbed for five minutes. The pellet is then broken into largechunks and the final cell suspension is placed into T75 flasks (2.5 mlper flask) into which 7.5 ml HT medium is present. The resulting cellsuspension is maintained at 37° C. to grow the fused cells. After 24hours, 10 ml of HT medium are admixed to the flasks followed six hourslater by admixture of 0.3 ml of 0.04 mM aminopterin. Forty-eight hoursafter fusion, 10 ml of HAT (hypoxanthine/aminopterin/thymidine) mediumare admixed to the flasks.

Three days after fusion, viable cells are plated out in 96-well tissueculture plates at about 2×10⁴ viable cells per well (768 total wells) inHAT buffer medium as described in Kennett et al., Curr. Top. Microbiol.Immunol., 81:77 (1978). The cells are fed seven days after fusion withHAT medium and at approximately four to five day intervals thereafter asneeded with HT medium. Growth is followed microscopically and culturesupernatants are collected about two weeks later. The culturesupernatants from HAT resistant cultures are subsequently assayed forthe presence of respective cortistatin-specific antibody by solid-phaseELISA as described in Example 3C and selected as hybridomas that producean antibody of this invention.

The anti-cortistatin antibodies are useful in detecting the presence ofcortistatin antigen in a human tissue or body sample through theformation of an immunoreaction complex as obtained in binding assays insitu, ELISA methodologies, by immunohistochemical methods includingtissue staining and flow cytometry, or by Western blot analysis. Inaddition, the anti-cortistatin antibodies prepared herein are useful inmethods to inhibit the physiological response of cortistatin receptorfollowing occupancy and activation by cortistatin as described below inExample 8.

4. Detection of Cortistatin Nucleic Acids

A. Detection of Cortistatin mRNA

To characterize cortistatin, the distribution of preprocortistatin mRNAwas assessed by Northern blot analysis. Total cellular RNA was preparedfrom homogenized tissues including rat brain, anterior pituitary,adrenal gland, liver, spleen, thymus, ovary and testes, according to themethod described by Chirgwin et al., Biochem., 18:5294-5299 (1979). TheRNA was enriched for poly(A)+ RNA by oligo(dt)-cellulose chromatographyas described by Aviv et al., Proc. Natl. Acad. Sci., USA, 69:1408-1412(1972). Two g of poly(A)+ selected RNA were fractionated by gelelectrophoresis and transferred to nitrocellulose. The latter was thenhybridized with a labeled cortistatin cDNA probe having the nucleotidesequence (SEQ ID NO 1). Hybridization conditions were essentially asdescribed by Forss-Petter et al., J. Mol. Neurosci., 1:63-75 (1989). Acyclophilin probe described by Danielson et al., DNA, 7:261-267 (1988)was hybridized to the same blot as a control for concentration andintegrity of the RNA samples.

The results of the mRNA screening are shown in FIG. 4. A single band ofapproximately 600 bp was detected in samples prepared from brain but notadrenal gland, liver, spleen, thymus, ovary, testes or anteriorpituitary. The result suggests that the rat preprocortistatin clonewhose sequence is shown in FIG. 1 is nearly full length.

Based on the limited distribution of preprocortistatin mRNA in brain,the cellular distribution of cortistatin mRNA was determined by in situhybridization (ISH) on rat brain sections on free-floating sections aspreviously described by deLecea et al., Mol. Brain Res.,. 25:286-296(1994). For double ISH, both probes (digoxigenin- and ³⁵S-labeled) wereincubated together on tissue sectins followed by washing and a secondincubation of the sections with an anti-digoxigenin Fab fragmentconjugated to alkaline phosphatase (1:3000; Boehringer Mannheim,Indianapolis, Ind.) as described by the manufacturer. For combined insitu hybridization-immunohistochemistry, in situ hybridization wasperformed first. Sections were then equilibrated in PBS, blocked with 4%BSA and incubated with a somatostatin antiserum described by Morrison etal., Brain Res., 262:344-351 (1983) (S309 diluted 1:2000). This antibodyrecognizes epitopes in the N-terminus of somatostatin-28 and thereforeis unlikely to crossreact with cortistatin-14. Cells containing morethan 20 silver grains over the cell body were considered positive, andwere counted as an average in eight different sections from threedifferent animals.

Signals were detected only in scattered cells throughout the cerebralcortex and hippocampus. Of note, no signal was detected in hypothalamus,an important site of somatostatin expression. In the cortex, signalswere especially abundant in layers II-III and VI. The visual/temporalcortex displayed about twice as many cortistatin-positive neurons as thesomatosensory cortex. In the hippocampus, cortistatin mRNA expressionwas found in some non-pyramidal cells in the subiculum and in thestratum oriens of the CA1 and CA3 fields, suggesting that cortistatinmRNA might be present in GABAergic interneurons.

To assess this aspect, double in situ hybridization was performed todetect coexpression of cortistatin and GAD₆₅/GAD₆₇ mRNAs, which encodethe synthetic enzymes for GABA as described by Erlander et al., Neuron,7:91-100 (1991). All cortistatin-expressing cells also contained eitherGAD₆₅ or GAD₆₇ mRNA, thus providing evidence for the GABAergic nature ofthese neurons.

By combined immunohistochemistry and in situ hybridization, cortistatinand somatostatin appear in different populations of interneurons; whilecortistatin is selectively expressed in cells of the cerebral cortex andhippocampus, somatostatin-containing neurons have a wider distribution.Moreover, cortistatin and somatostatin can exist in differentpopulations of interneurons, as demonstrated by the limitedco-localization of somatostatin immunoreactivity and cortistatin mRNA.In cortical layers II-III, most positive interneurons expressed eithersomatostatin or cortistatin but not both, although in some othercortical areas. (e.g., layer VI of the visual cortex) as many as 40% ofcortistatin-positive cells also contained somatostatin-likeimmunoreactivity. In the hippocampal CA fields, most (>80%)cortistatin-positive cells were also positive for somatostatinimmunoreactivity. Cortistatin was not present in the hilus, which wasrich in somatostatin-positive cells.

Hybridization to northern blots of mouse tissues revealed the presenceof two bands in brain but not liver, kidney or thymus. Two bands werealso observed in the human brain sample.

B. Detection of CTG Repeats in Cortistatin

As previously described in Example 1, the rat, human and mousepreprocortistatin cDNAs are characterized by having multiple iterationsof the triplet CTG in the nucleotide sequence encoding leucine residuesin a portion of the signal sequence. For the rat sequence, sixiterations of the trinucleotide CTG repeats are present while in themouse sequence three CTG repeats are present for encoding leucine.Similarly, for the human sequence four iterations of the CTG repeats arepresent.

As previously mentioned, triplet expansions have been determined to bethe genetic basis for neurological diseases, e.g., myotonic dystrophy asdescribed by Brook et al., Cell, 68:799-808 (1992) and fragile-Xsyndrome as described by Fu et al., Cell, 67:1047-1058 (1991). Inmyotonic dystrophy patients who are mildly affected, at least 50 CTGrepeats are present. In severely affected individuals, the expansion canexist up to several kilobase pairs. In contrast, in the normalpopulation, the repeat sequence is highly variable ranging from 5 to 27copies. Individuals with varying severities of fragile-X have beensimilarly characterized.

Thus, screening for the presence of a region of DNA in which the repeatsare present in either normal, underexpansion or overexpansion form canprovide a genetic basis for diagnosis for some diseases. The same may betrue for cortistatin in that expansion of the region may contribute tothe basis for a sleep-related or disease related to cortical activity ofthe brain. Consequently, one aspect of the present invention is agenetic screening method to determine the nature of the lysine-encodingtriplets in a specified region of cortistatin nucleotide sequence. Byscreening a large number of samples from a population of normalindividuals along with those having sleep disturbances or disorders, therange for normal variability can be determined as well as thecorrelation of repeat length with a disorder and severity thereof.

Therefore, in view of the nucleotide sequence similarity between rat andmouse preprocortistatin as respectively shown in FIGS. 1 and 3,oligonucleotide primer sequences have been designed to allow forpositional amplification by polymerase chain reaction (PCR) of targetnucleic acid samples. For screening for transcribed cortistatin, thenucleic acid sample is derived from brain tissue biopsy. Alternatively,the designed primers are useful for amplifying genomic DNA obtained fromreadily available cells, such as peripheral blood leucocytes.

The 5′ primers for rat and mouse respectively encode the amino acidresidue sequence Gly-Lys-Arg-Pro-Ser-Ala (SEQ ID NO 17) andGly-Lys-Trp-Pro-Ser-Ala (SEQ ID NO 18). The nucleotide sequence for the3′ primer is the same for rat and mouse sequence encoding the amino acidresidue sequence Trp-Trp-His-Glu-Trp-Ala (SEQ ID NO 19) as written inamino terminal to carboxy terminal direction of the cortistatin proteinas shown in FIGS. 1 and 3 and in the respective SEQ ID NOs 2 and 5.Preferred nucleotide sequences for primers corresponding to the aminoacids in SEQ ID NOs 17, 18 and 19 are respectively 5′GGCAAGCGGCCGTCAGCC3′ (SEQ ID NO 20), 5′ GGCAAGTGGTCAGCC (SEQ ID NO 21)and 5′ AGACTCATGCCACCA3′ (SEQ ID NO 22).

PCR amplifications are then performed with a sample of nucleic acidaccording to methods well known to one of ordinary skill in the art andas described in “Current Protocols in Molecular Biology”, Ausubel etal., eds, Chapter 15, Wiley & Sons, Inc., New York (1989).

The resultant amplified DNA is then analyzed by gel electrophoresis forthe presence of triplet expansions in the region between the two primerpairs, the region of which for rat and mouse are respectively 171 and159 nucleotides in length. The amplified rat region corresponds tonucleotide positions 51 through 221 in SEQ ID NO 1 and for mouse thecorresponding nucleotide positions in SEQ ID NO 4 are from 49 through207. An increase in the molecular weight of the fragment is indicativeof expansion of a selected triplet repeat. To confirm the presence ofexpansion and/or compression of the region, the PCR fragments aresequenced. Appropriate diagnoses are then readily made.

5. Detection of Cortistatin Protein

A. Receptor Binding Assays

To determine the binding specificity of cortistatin and in view of itssimilarity to somatostatin, assays to assess the binding of chemicallysynthesized, linear cortistatin-14 peptide prepared in Example 2 tosomatostatin receptors on GH₄ pituitary cells were performed.

GH₄ cells, obtained from Dr. Kaare M. Gautvik (University of Oslo) orATCC, were grown in MEM medium with 12% fetal calf serum in 6-wellplates at 10⁶ cells/ml. Each well then received 10⁶ cpm/ml of ¹²⁵Isomatostatin-14 (NEN, DuPont, Wilmington, Del.) alone or with increasingconcentrations of somatostatin-14 peptide (Sigma) or cortistatin-14peptide (95%, purified by reverse phase HPLC ranging from 10⁻¹⁰ M to10⁻⁶ M. Aprotinin and leupeptin (Sigma; 2 μg/ml) was included as itreduced non-specific binding to 20% of total bound radioactivity. Thebinding of ¹²⁵I somatostatin in the presence of 10⁻⁷ M somatostatin wasconsidered as unspecific binding as described by Schonbrunn et al., J.Biol. Chem., 235:6473-6483 (1978).

As shown in FIG. 5A, both cortistatin-14 (filled circles) andsomatostatin-14 (empty circles) effectively displaced¹²⁵I-somatostatin-14 binding in a dose-dependent manner, with anestimated K_(d) of 5×10⁻⁹ M, very close to that previously reported forsomatostatin as described by Schonbrunn et al., J. Biol. Chem.,235:6473-6483 (1978). The combined data from four independentexperiments are plotted as mean values ± standard error. As a control,thryoid releasing hormone (TRH) (Calbiochem, La Jolla, Calif.) andvasoactive intestinal peptide (VIP) (Bachem, Switzerland) did not showany displacement of ¹²⁵I-somatostatin.

Parallel assays are readily performed with the cortistatin proteins andpolypeptides listed in Table 1, prepared by either recombinant orsynthetic means as described above.

Comparable binding assays, as described in Example 6, are performed withGH₄ cells or cortistatin-specific positive cells or tissues containingsuch to identify a cortistatin analog, also referred to as a cortistatinreceptor ligand.

B. Receptor Activation Assays

To investigate whether cortistatin modulates somatostatin receptoractivation, the concentration of cyclic AMP (cAMP) was determinedfollowing stimulation of GH₄ cells with VIP or TRH in the presence orabsence of either cortistatin or somatostatin.

For the cAMP assays, GH₄ cells were grown under the same conditions asfor the binding assays. The cells were washed with MEM without serum,but containing leupeptin, aprotinin and 1 mM 3-isobutyl-methyl-xantine(IBMX). The cells were pretreated with somatostatin-14 andcortistatin-14 for 15 minutes before VIP at 10⁻⁶ M was added or TRH at10⁻⁷ M. To each well, ³H cyclic AMP was added before the content wasremoved, to calculate recovery. For cAMP measurements, a RIA kit(Amersham, Arlington Heights, Ill.) was used according to theinstructions of the manufacturer. Each time point represents 2-4replicates and the experiments were carried out twice.

As shown in FIG. 5B, both VIP and TRH at respective concentrations of10⁻⁶ M and 10⁻⁷ M increased the intracellular concentration of cAMPwhile the somatostatin and cortistatin peptides at 10⁻⁶ M had no effectas compared to control.

As shown in FIG. 5C, both statin peptides showed indistinguishablyeffective inhibition of VIP and TRH stimulation of cAMP in cells. Bothpeptides at 10⁻⁸ M completely inhibited the action of TRH, whereas theyattenuated the effect of VIP in a dose-dependent manner by about 50% at10⁻⁶ M.

Therefore, in view of the results shown in FIGS. 5A-5C, cortistatinappears to act as an agonist on the endogenous somatostatin receptorsexpressed by GH₄ cells, although these cells may express a heterogeneouspopulation of receptors and the agonist activity may not necessarily beits role at its sites of expression. Although a cortistatin-specificreceptor has yet been identified, the assays described in Example 7using the cortistatin proteins and polypeptides listed in Table 1,prepared by either recombinant or synthetic means are designed tofacilitate such identification of a cortistatin-specific receptor.

6. Detection of Cortistatin Analogs and Antagonists

The receptor binding and cAMP activation assays described in Example 5are used in this invention to screen for cortistatin analogs as well asantagonists, the latter of which includes cortistatin-specificantibodies. Anti-cortistatin antibodies that are cortistatin antagonistsare also referred to as cortistatin receptor antagonists in that theantibody blocks the binding of the cortistatin ligand to its receptor,thereby preventing receptor occupancy and activation.

In the receptor binding assay, analogs of cortistatin are identified inthe same manner as was used to identify cortistatin as an agonist ofsomatostatin receptors. In a parallel assay, a cortistatin antagonist,such as an anti-cortistatin peptide antibody preparation as described inExample 4, is identified by blocking the cortistatin-14 ability todisplace labeled somatostatin from binding to thesomatostatin-receptor-bearing cells or to cortistatin-receptor-bearingcells. In a modified receptor binding assay using ¹²⁵I-cortistatin-14 orany labeled cortistatin protein or polypeptide of this invention,preferably procortistatin, a cortistatin antagonist can be identified ifthe candidate molecule is shown to displace the binding of the labeledcortistatin peptide to GH4 cells or cells bearing cortistatin-specificreceptors.

Confirmation of a cortistatin antagonist is provided in the cAMP assaysby incubation of the molecule with the cortistatin-14 peptide prior toaddition of the cAMP activators. A cortistatin antagonist is identifiedby the measured inhibition of cAMP accumulation in assays performed asdescribed for the data shown in FIG. 5C. Cortistatin analogs areidentified by the effective inhibition of cAMP accumulation in parallelto that shown in FIG. 5C.

Further confirmation of the identification of a cortistatin analog orantagonist is provided in the in vitro and in vivo electrophysiologicalassays as described in Example 8 where the functional physiologicalresponses by cortistatin are distinct from those elicited bysomatostatin, the responses of which are dependent upon specificreceptor activation.

Cortistatin agonists are also identified by their binding patterns onrat brain cryosections, as has been described for somatostatin byPelletier et al., Meth. Enzymol., 124:607-615 (1986), the disclosure ofwhich is hereby incorporated by reference.

7. Detection of Cortistatin-Specific Receptor

For identifying a cortistatin-specific receptor in the brain,cortistatin-specific binding sites are detected as described by Tran etal., Eur. J. Pharmacol., 101:307-309 (1984), the disclosure of which ishereby incorporated by reference. Briefly, synthetic analogs ofcortistatin presented in single-letter code, (YPCKNFFWKTFSSCK (SEQ ID NO23) or PCKNFFYKTFSSCK (SEQ ID NO 24)), are labeled with ¹²⁵I severalmethods and purified by reverse phase HPLC. Different amounts of¹²⁵I-labeled cortistatin analogs are then incubated with rat braincryosections in the presence of 10⁻⁶ M cold competitive analog.

Alternatively, tritiated analogs are synthesized and used for in situautoradiography.

Cortistatin-specific receptors are also identified by binding labeledcortistatin analogs, that include cortistatin proteins and polypeptideslisted in Table 1, prepared by either recombinant or synthetic means asdescribed above, to biochemical membrane preparations from cortex.Synthetic cortistatin is immobilized to activated agarose columns (suchas BioGel 10 columns from BioRad) and used to purify cortistatin-bindingproteins from brain extracts. Such methods are well known in the art.

8. Physiology of Cortistatin and Therapeutic Methods

Somatostatin is known to hyperpolarize central neurons by increasingpotassium conductances as described by Inoue et al., J. Physiol.,407:177-198 (1988), including the voltage-dependent potassium M current(see Moore et al., Science, 239:278-280 (1988) and Jacquin et al., Proc.Natl. Acad. Sci., USA, 85:948-952 (1988)). To complete thecharacterization of the physiological role of cortistatin, assays wereperformed to determine whether cortistatin had somatostatin-like effectson hippocampal neurons by means of current- and voltage- clamprecordings in the hippocampal slice preparation.

Intracellular recordings were obtained in rat hippocampal slices usingsharp glass micropipettes as previously described by Schweitzer et al.,J. Neurosci., 13:2033-2049 (1993). Recordings were made from 11hippocampal CA1 pyramidal cells with an average resting membranepotential of −70±1 mV (mean ± s.e.m.) and action potential of 103±2 mV.Current-clamp recording of a CA1 neuron manually depolarized to −65 mV(resting membrane potential was −70 mV) to elicit action potentialfiring (upward deflections, truncated) is shown in FIG. 6A.

Superfusion of 1 μM cortistatin-14 peptide (bar above record), preparedas described in Example 3, like somatostatin-14, hyperpolarized theseneurons (10 of 11 cells), in association with inhibition of actionpotential firing, followed by recovery to control levels upon washout ofthe peptide. Unlike somatostatin, the cortistatin-14-mediated effectdeveloped slowly, reaching a maximum steady effect six to eight minutesafter the onset of the response. This contrasted with the time-to-peakof somatostatin's effect on these neurons under the same experimentalconditions that was much shorter (2-3 minutes).

To determine the mechanism of the cortistatin-induced inhibition, weassessed the effect of cortistatin on the M current (I_(M)), anon-inactivating voltage-dependent potassium current seen in hippocampalneurons as described by Halliwell et al., Brain Res., 250:71-92 (1982).

In voltage clamp assays, the I_(M) relaxation was observed by applyinghyperpolarizing steps (5 to 25 mV) from a holding potential of −43 to−48 mV. FIG. 6B shows the voltage-clamp recording of a CA1 neuron heldat −43 mV; an I_(M) relaxation was evoked with 10 mV hyperpolarizingstep.

As previously described for somatostatin (Moore et al., Science,239:278-280 (1988) and Schweitzer et al., Nature, 346:464-466 (1990)),cortistatin-14 (1 μM, 7 minutes) superfusion increased the amplitude ofthe I_(M) relaxation by 60% (see arrows) concomitantly with an outwardsteady-state current, as shown in FIG. 6B with the dotted line ascontrol holding current, with recovery to control levels upon washout.

The inhibitory effects of cortistatin on the excitability of CA1pyramidal neurons as viewed by population spike (PS) amplitudes wasparalleled in vivo in the anesthetized preparation. For the in vivostudies, male Sprague-Dawley rats were anesthetized with halothane(0.9-1.1%). The commisural pathway was stimulated and elicited fieldpotentials in the CA1 region essentially as described by Steffensen etal., Brain Res., 538:46-53 (1991). Cortistatin (1 mg/ml) was dissolvedin saline and administered iontophoretically through one barrel of amultibarreled micropipette. As shown in FIG. 6C, stimulus-responsecurves were generated and the PS amplitude was monotonically related tostimulus intensity tested at three response levels: threshold,half-maximal and maximal (control mean half-maximal PS amplitude=4.7 mV±0.5; n=5). Asterisks represent significance levels at P<0.05. The datawas compiled with software (LabView Instruments, National Instruments,Austin, Tex.) as described by Steffensen et al., Brain Res., 538:46-53(1991).

Stimulation of the monosynaptic afferent input to the CA1 region evokeda characteristic population spike (PS) that represents the synchronousfiring of pyramidal cells, superimposed on a field synaptic potentialwaveform as described by Anderson et al., Exp. Brain Res., 13:208-221(1971). As is apparent in FIG. 6C, microiontophoretic application ofcortistatin, like somatostatin, significantly decreased PS amplitudesboth at half-maximal and maximal levels of stimulation.

As cortistatin is expressed in interneurons located in the cerebralcortex and hippocampus, its effects on cortical measures of neuronalexcitability was next determined in vivo. Up to 6 nmoles ofHPLC-purified synthetic cortistatin-14 peptide prepared in Example 3were infused into the brain ventricles of rats (n=5) and recorded theelectroencephalogram (EEG) for four hours after peptide injection. Inaddition, rats were observed for changes in spontaneous behavior througha one-way window. Standard methodologies for chronicelectrophysiological preparation and data acquisition were used.Procedures for EEG recordings and PP studies are as described byProspero-Garcia et al., Pharmacol. Biochem. Behav., 49:413 (1994) andSteffensen et al., Brain Res., 652:149 (1994).

FIGS. 7A-1, 7A-2, 7A-3 and 7A-4 show the effects of theintracerebroventricular administration of cortistatin on the sleep-wakecycle of the rat. FIGS. 7A-1, 7A-2, 7A-3 and 7A-4 respectivelyillustrate wakefulness, slow-wave sleep 1, slow-wave sleep 2 and REMsleep. The effects of the sleep cycle were assessed with cortistatin-14dosages at 100 ng, 500 ng, 1 μg and 10 μg as plotted from left to rightin the bar graphs as shown against saline control. Asterisks representsignificance levels of P<0.05; ANOVA.

Cortistatin-14-peptide-treated animals demonstrated a clear hypoactivebehavior compared to the saline-injected rats, but kept their eyes openand displayed other signs of wakefulness for a short period of time(15-20 minutes). In these animals, the EEG showed a dramatic increase incortical slow waves (1-4 Hz). As shown by referring to FIGS. 7A-1through 7A-4, polygraphic monitoring of arousal states subsequent to theadministration of cortistatin also indicated that rats spent up to 75%of the four hour recording time in slow-wave sleep compared to 40% insaline-treated control animals. A significant reduction on paradoxical(REM) sleep was also observed with the highest dose of cortistatin, inclear contrast to the reported increase in REM sleep afteradministration of a similar dose of somatostatin as described by Danguiret al., Brain Res., 367:26-30 (1986).

In addition, two doses of cortistatin (0.1 and 1 μg) were tested inreversed sleep cycle rats (n=7/group). Since in this model the animalshave already achieved their physiological demand of sleep as describedby Inoue et al., Neurosci. Lett., 49:207-209 (1984), the efficiency ofcortistatin as a sleep-inducing molecule would be more evident.Cortistatin significantly decreased wakefulness (49.1±2%. insaline-treated control rats; 31±2.6% with 100 ng of cortistatin;32±8.78% with 1 μg) and increased slow wave sleep 2 (35.8±1.8% incontrols; 51.5±3.8% with 100 ng; 53.7±7.3% with 1 μg) but did notsignificantly affect slow wave sleep 1 (8.8±1.7% in control rats;8.1±0.9% with 100 ng; 5.6±1.4% for 1 μg) or REM sleep (6.2±1.4% incontrols; 9.3±1.7% with 100 ng; 7.7±1.3% with 1 μg). These resultsstrongly support the discovery of the present invention that cortistatinis a sleep-modulating substance.

To investigate the mechanisms by which cortistatin might facilitatecortical slow waves and reduce the duration of REM sleep, thepossibility that cortistatin produced these effects, in part, bymodulating acetylcholine (ACh) activity as described by Steriade et al.,Ann. Rev. Pharmacol. Toxicol., 27:137-156 (1987) was investigated. Theinteractions between cortistatin and ACh on hippocampal CA1 neurons wasexamined using an evoked paired-pulse (PP) stimulation paradigm to testfeed-forward and feed-back inhibitory processes mediated in part byhippocampal interneurons (see Andersen et al., J. Neurophysiol.,27:607-619 (1964) and Kandel et al., J. Neurophysiol., 24:243-259(1961).

The effects of iontophoretically applied ACh (0.9 M), somatostatin (1.5mM) and cortistatin on PP responses in CA1 neurons in vivo are shown inFIG. 7B. Stimulation intensity was adjusted after drug treatment toachieve control PS amplitudes before performing PP studies.

Without ACh, stimulation at half-maximal stimulus levels revealed acharacteristic biphasic PP response curve as described by Steffensen etal., Brain Res., 538:46-53 (1991). Microiontophoretic application of AChsignificantly antagonized the typical inhibitory phase of PP responsesseen at interstimulus intervals from 60-80 msec (asterisks correspond toa significance level of P<0.05; see FIG. 7B; data from 80 msec intervalsare shown in FIGS. 7C-E). FIGS. 7C-7F each representative recordings offield potentials elicited in CA1 by commisural stimulation. In thebaseline recordings shown in FIG. 7C (calibration bars: 2 mV and 10 ms),the second response is completely inhibited at this interstimulusinterval (80 ms). Iontophoretic administration of ACh reduced PPinhibition as shown in FIG. 7D. This effect was antagonized by thesimultaneous application of cortistatin shown in FIG. 7E. In FIG. 7F,somatostatin markedly decreased PP inhibition (calibration bar 1 mV and10 ms).

To summarize, cortistatin itself (not shown) had no significant effectson PP responses in CA1 but as shown in FIG. 7E, completely antagonizedthe effects of ACh (P<0.05). The effects of somatostatin on PP responseswere similar to those of ACh (FIG. 7B; data from 80 msec shown in FIG.7F) but were significantly different from those of cortisatin (asteriskscorrespond to significance levels of P<0.05).

To investigate further whether cortistatin also interacts withcholinergic systems that regulate cortical function, the effects ofcortistatin on ACh-induced desynchronization of EEG in the cerebralcortex of anesthetized rats was determined. Thus, ACh and cortistatinwas microiontophoretically (100-250 nA) applied on local EEG activityrecorded in the visual cortex. For the data shown in FIG. 7G, theresults from the experimental groups were derived from averaged EEGspectral activity determinations and expressed as means I± s.e.m.(Asterisk represents significance levels P<0.05 from baseline; twoasterisks represent significance levels P<0.05 from ACh; ANOVA).

As shown in FIG. 7G, electrophoretic application of ACh markedlydesynchronized the local EEG by increasing the potency of the theta(4.5-8 Hz) and beta (13-20 Hz) bands, i.e., increased the averaged EEGpower spectra (four second epochs over three minutes) in the 8-16 Hzfrequency band range. In contrast, the averaged EEG during infusion ofcortistatin alone or ACh and cortistatin combined were not differentfrom baseline recordings of slow waves of 0.5-4 Hz. The effect of Achwas effectively prevented by the simultaneous electrophoreticapplication of cortistatin-14 peptide that antagonized the increase infast frequency activity produced by Ach alone.

To further the diagnostic and therapeutic methods of this invention, theelectrophysiological assays described herein are also performed with theother cortistatin proteins and polypeptides listed in Table 1, preparedby either recombinant or synthetic means as described above. Inaddition, the anti-cortistatin antibodies of this invention are usefulin in vitro assays as well as in vivo applications as described above tofacilitate the therapeutic amelioration of a sleep disorder, such asnarcolepsy, as the anti-cortistatin antibodies prevent activation ofcortistatin receptor by binding cortistatin.

9. Analysis of Structural and Functional Characteristics of Cortistatinwith Somatostatin

The present invention describes the discovery and isolation of a cDNAclone of a mRNA that encodes the precursor of a novel member. of thesomatostatin family whose distribution is primarily restricted toGABAergic cortical interneurons. GABAergic neurons have been shown tofinely modulate the output of principal neurons of the cerebral cortexand hippocampus as described by Buhl et al., Nature, 368:823-828 (1994),areas that have been implicated in arousal state and complex cognitivefunctions, including learning and memory (see Wilson et al., Science,265:676-679 (1994). Cortistatin may therefore play a role in elaboratingthese functions.

The peptides, cortistatin-14 and somatostatin-14, appear to have similareffects on the physiology of hippocampal neurons. As shown in theExamples above, both peptides bind to somatostatin receptors on GH₄cells with very similar affinities, inhibit the hormonally-inducedaccumulation of cAMP in these cells with similar efficiencies, causeneuronal hyperpolarization and increase the M-current in hippocampalneurons, thus suggesting that they could act through the same receptorsin vivo. The presence of the common amino acid residues FWKT (SEQ ID NO2 from position 104 to 107), that have been shown to be critical forsomatostatin binding, also supports the idea of cortistatin binding tosomatostatin receptors.

Nevertheless, cortistatin's effects on the activity of hippocampalneurons in vivo and on sleep physiology are clearly distinct from thoseof somatostatin. Thus, cortistatin could differentially bind tosomatostatin receptor subtypes different from the ones analyzed here, orit could act on μ-opioid receptors, as has been demonstrated for thesomatostatin analog octeotride and the μ receptor antagonist CTOP asdescribed by Maurer et al., Proc. Natl. Acad. Sci., USA, 79:4815-4817(1982). Cortistatin and somatostatin co-exist in some interneurons inthe deep cortical layers and hippocampus, thus suggesting that thesestatins may compete for the same receptors, or they may be released fromdifferent synaptic boutons in response to different stimuli.

Cortistatin appears to be an inhibitory neuromodulator in thehippocampus. The hyperpolarization seen in current-clamp recordings islikely to be due, at least in part, to the augmentation of thenon-inactivating potassium M-current. However, as with somatostatin,another K⁺ channel mechanism could also participate in thecortistatin-induced inhibitory effect (see Schweitzer et al., J.Neurosci., 13:2033-2049 (1993). Indeed the delayed effects seen in thehippocampal slice, along with the observed differences in functionalresponses, may suggest a distinct, uncharacterized cortistatin receptor.In binding studies to cloned somatostatin receptors, cortistatinexhibited affinity comparable to somatostatin for somatostatin receptor1 (SSTR1), but far lesser affinity for SSTR 2-5. Since SSTR3 and 4 arethe predominant SSTRs in the brain regions in which cortistatin isexpressed, the existence of an uncharacterized receptor is favored.Alternatively, the delayed effects could merely indicate a more aviduptake, degradation or more limited access to slice tissue due to anadditional positive charge present in cortistatin, compared tosomatostatin. These factors could require longer-term saturation andprolonged superfusion before significant receptor activation couldoccur.

The administration of cortistatin into the brain ventricles induced amarked enhancement of slow-wave sleep and decreased the REM phase in adose-dependent manner. These effects are opposite to those described forsomatostatin, which facilitates REM sleep generation as described byDanguir et al., Brain Res., 367:26-30 (1986). Moreover, the behaviorexhibited by cortistatin-treated rats was clearly different from thecharacteristic behaviors (i.e. hypermotility, barrel rotation)previously described for somatostatin-treated rats (see, Rezek et al.,Pharmacol. Biochem. Behavior, 5:73-77 (1976) and Vecsei et al.,Peptides, 10:1153-1157 (1989). The data provided herein indicating thatcortistatin enhances slow-wave sleep and reduces REM sleep suggest thatthe effects of cortistatin are produced, in part, by modulatingcholinergic function.

Induction of slow-wave sleep is characterized by the appearance of slowfrequency waves in cortical activity and the reduction of AChavailability in the cerebral cortex, as shown in several preparations(see Kodama et al., 1Neurosci. Lett., 114:277-282 (1990) and Marrosu etal., Brain Res., 671:329-332 (1995). Recent electrophysiological studiesin vivo have shown that the expression of slow-wave sleep in the ratcerebral cortex coincides with an increase in paired-pulse inhibition inthe hippocampal CA1 region as described by Prospero-Garcia et al.,Neurosci. Lett., 156:158-162 (1993). By contrast, during activewakefulness and REM sleep there is a marked reduction in PP inhibition,when the availability of ACh is much higher. The lack of effect ofcortistatin on PP inhibition contrasts with the potent inhibitory effectof somatostatin on this measure observed in the present invention, whichis supported by the somatostatin reduction of GABA-mediated synapticpotentials in CA1 pyramidal neurons previously reported by Scharfman etal., Brain Res., 493:205-211 (1989).

Together, this provides another functional difference betweencortistatin and somatostatin. These results are consistent with those inreverse-phased and awake rats, where cortistatin markedly reduced theduration of cortical electrical activity that is associated with thecholinergic system, as well as the ACh-induced desynchronization oflocal EEG. Therefore, cortistatin antagonizes the effects of ACh in boththe hippocampus and the cerebral cortex in vivo. The findings reportedhere thus provide the physiological basis that this novel neuropeptide,cortistatin, functions as a regulator of neuronal activity and sleep. Assuch, cortistatin, analogs and antagonists thereof, are valuablereagents in use in the diagnostic and therapeutic methods of thisinvention.

The foregoing specification, including the specific embodiments andexamples, is intended to be illustrative of the present invention and isnot to be taken as limiting. Numerous other variations and modificationscan be effected without departing from the true spirit and scope of theinvention.

26 438 base pairs nucleic acid single linear cDNA NO NO CDS 30..368 1AAAGCACAGA CTTCAGGTTT CCAAGGAGG ATG GGT GGC TGC AGC ACA AGA GGC 53 MetGly Gly Cys Ser Thr Arg Gly 1 5 AAG CGG CCG TCA GCC CTC AGT CTG CTG CTGCTG CTG CTG CTC TCG GGG 101 Lys Arg Pro Ser Ala Leu Ser Leu Leu Leu LeuLeu Leu Leu Ser Gly 10 15 20 ATC GCA GCC TCT GCC CTC CCC CTG GAG AGC GGTCCC ACC GGC CAG GAC 149 Ile Ala Ala Ser Ala Leu Pro Leu Glu Ser Gly ProThr Gly Gln Asp 25 30 35 40 AGT GTG CAG GAT GCC ACA GGC GGG AGG AGG ACCGGC CTT CTG ACT TTC 197 Ser Val Gln Asp Ala Thr Gly Gly Arg Arg Thr GlyLeu Leu Thr Phe 45 50 55 CTT GCC TGG TGG CAT GAG TGG GCT TCC CAA GAC AGCTCC AGC ACC GCT 245 Leu Ala Trp Trp His Glu Trp Ala Ser Gln Asp Ser SerSer Thr Ala 60 65 70 TTC GAA GGG GGT ACC CCG GAG CTG TCT AAG CGG CAG GAAAGA CCA CCC 293 Phe Glu Gly Gly Thr Pro Glu Leu Ser Lys Arg Gln Glu ArgPro Pro 75 80 85 CTC CAG CAG CCC CCA CAC CGG GAT AAA AAG CCC TGC AAG AACTTC TTC 341 Leu Gln Gln Pro Pro His Arg Asp Lys Lys Pro Cys Lys Asn PhePhe 90 95 100 TGG AAA ACC TTC TCC TCG TGC AAG TAGCCCGAGC CTGACCGGAGCCTGACCGGC 395 Trp Lys Thr Phe Ser Ser Cys Lys 105 110 CACCCTGTGAATGCAGCCGT GGCCTGAATA AAGAGTGTCA AGT 438 112 amino acids amino acidlinear protein 2 Met Gly Gly Cys Ser Thr Arg Gly Lys Arg Pro Ser Ala LeuSer Leu 1 5 10 15 Leu Leu Leu Leu Leu Leu Ser Gly Ile Ala Ala Ser AlaLeu Pro Leu 20 25 30 Glu Ser Gly Pro Thr Gly Gln Asp Ser Val Gln Asp AlaThr Gly Gly 35 40 45 Arg Arg Thr Gly Leu Leu Thr Phe Leu Ala Trp Trp HisGlu Trp Ala 50 55 60 Ser Gln Asp Ser Ser Ser Thr Ala Phe Glu Gly Gly ThrPro Glu Leu 65 70 75 80 Ser Lys Arg Gln Glu Arg Pro Pro Leu Gln Gln ProPro His Arg Asp 85 90 95 Lys Lys Pro Cys Lys Asn Phe Phe Trp Lys Thr PheSer Ser Cys Lys 100 105 110 110 amino acids amino acid linear proteinC-terminal 3 Gln Cys Ala Leu Ala Ala Leu Cys Ile Val Leu Ala Leu Gly GlyVal 1 5 10 15 Thr Gly Ala Pro Ser Asp Pro Arg Leu Arg Gln Phe Leu GlnLys Ser 20 25 30 Leu Ala Ala Ala Thr Gly Lys Gln Glu Leu Ala Lys Tyr PheLeu Ala 35 40 45 Glu Leu Leu Ser Glu Pro Asn Gln Thr Glu Asn Asp Ala LeuGlu Pro 50 55 60 Glu Asp Leu Pro Gln Ala Ala Glu Gln Asp Glu Met Arg LeuGlu Leu 65 70 75 80 Gln Arg Ser Ala Asn Ser Asn Pro Ala Met Ala Pro ArgGlu Arg Lys 85 90 95 Ala Gly Cys Lys Asn Phe Phe Trp Lys Thr Phe Thr SerCys 100 105 110 427 base pairs nucleic acid single linear cDNA NO NO CDS25..354 4 GCACGAGGCT CAGCACGTCC GAGG ATG ATG GGT GGC CGA GGC ACA GGA GGC51 Met Met Gly Gly Arg Gly Thr Gly Gly 1 5 AAG TGG CCC TCA GCC TTC GGGCTG CTG CTG CTC TGG GGG GTC GCA GCC 99 Lys Trp Pro Ser Ala Phe Gly LeuLeu Leu Leu Trp Gly Val Ala Ala 10 15 20 25 TCC GCC CTT CCC CTG GAG AGTGGC CCT ACT GGC CAG GAC AGT GTG CAG 147 Ser Ala Leu Pro Leu Glu Ser GlyPro Thr Gly Gln Asp Ser Val Gln 30 35 40 GAA GCC ACC GAG GGG AGG AGC GGCCTT CTG ACT TTC CTT GCC TGG TGG 195 Glu Ala Thr Glu Gly Arg Ser Gly LeuLeu Thr Phe Leu Ala Trp Trp 45 50 55 CAC GAG TGG GCT TCC CAA GCC AGC TCCAGC ACC CCC GTC GGA GGG GGT 243 His Glu Trp Ala Ser Gln Ala Ser Ser SerThr Pro Val Gly Gly Gly 60 65 70 ACC CCC GGG CTG TCC AAG AGC CAG GAA AGGCCA CCC CCC CAA CAG CCC 291 Thr Pro Gly Leu Ser Lys Ser Gln Glu Arg ProPro Pro Gln Gln Pro 75 80 85 CCA CAC CTG GAT AAA AAG CCC TGC AAG AAC TTCTTC TGG AAA ACC TTC 339 Pro His Leu Asp Lys Lys Pro Cys Lys Asn Phe PheTrp Lys Thr Phe 90 95 100 105 TCC TCG TGC AAG TAACCCCACC CTGGGCATAGCACCCTGGCC ACCCTGTGAG 391 Ser Ser Cys Lys 110 ATGCCAACGA GACCTGAATAAAGACTGTCA ATCAAC 427 109 amino acids amino acid linear protein 5 MetMet Gly Gly Arg Gly Thr Gly Gly Lys Trp Pro Ser Ala Phe Gly 1 5 10 15Leu Leu Leu Leu Trp Gly Val Ala Ala Ser Ala Leu Pro Leu Glu Ser 20 25 30Gly Pro Thr Gly Gln Asp Ser Val Gln Glu Ala Thr Glu Gly Arg Ser 35 40 45Gly Leu Leu Thr Phe Leu Ala Trp Trp His Glu Trp Ala Ser Gln Ala 50 55 60Ser Ser Ser Thr Pro Val Gly Gly Gly Thr Pro Gly Leu Ser Lys Ser 65 70 7580 Gln Glu Arg Pro Pro Pro Gln Gln Pro Pro His Leu Asp Lys Lys Pro 85 9095 Cys Lys Asn Phe Phe Trp Lys Thr Phe Ser Ser Cys Lys 100 105 85 aminoacids amino acid linear protein C-terminal 6 Ser Ala Leu Pro Leu Glu SerGly Pro Thr Gly Gln Asp Ser Val Gln 1 5 10 15 Asp Ala Thr Gly Gly ArgArg Thr Gly Leu Leu Thr Phe Leu Ala Trp 20 25 30 Trp His Glu Trp Ala SerGln Asp Ser Ser Ser Thr Ala Phe Glu Gly 35 40 45 Gly Thr Pro Glu Leu SerLys Arg Gln Glu Arg Pro Pro Leu Gln Gln 50 55 60 Pro Pro His Arg Asp LysLys Pro Cys Lys Asn Phe Phe Trp Lys Thr 65 70 75 80 Phe Ser Ser Cys Lys85 29 amino acids amino acid linear protein C-terminal 7 Gln Glu Arg ProPro Leu Gln Gln Pro Pro His Arg Asp Lys Lys Pro 1 5 10 15 Cys Lys AsnPhe Phe Trp Lys Thr Phe Ser Ser Cys Lys 20 25 14 amino acids amino acidlinear protein C-terminal 8 Pro Cys Lys Asn Phe Phe Trp Lys Thr Phe SerSer Cys Lys 1 5 10 13 amino acids amino acid linear protein internal 9Gln Glu Arg Pro Pro Leu Gln Gln Pro Pro His Arg Asp 1 5 10 84 aminoacids amino acid linear protein C-terminal 10 Ser Ala Leu Pro Leu GluSer Gly Pro Thr Gly Gln Asp Ser Val Gln 1 5 10 15 Glu Ala Thr Glu GlyArg Ser Gly Leu Leu Thr Phe Leu Ala Trp Trp 20 25 30 His Glu Trp Ala SerGln Ala Ser Ser Ser Thr Pro Val Gly Gly Gly 35 40 45 Thr Pro Gly Leu SerLys Ser Gln Glu Arg Pro Pro Pro Gln Gln Pro 50 55 60 Pro His Leu Asp LysLys Pro Cys Lys Asn Phe Phe Trp Lys Thr Phe 65 70 75 80 Ser Ser Cys Lys29 amino acids amino acid linear protein C-terminal 11 Gln Glu Arg ProPro Pro Gln Gln Pro Pro His Leu Asp Lys Lys Pro 1 5 10 15 Cys Lys AsnPhe Phe Trp Lys Thr Phe Ser Ser Cys Lys 20 25 13 amino acids amino acidlinear protein internal 12 Gln Glu Arg Pro Pro Pro Gln Gln Pro Pro HisLeu Asp 1 5 10 31 base pairs nucleic acid single linear cDNA NO NO 13ATCGAGATCT AAGGAGGATG GGTGGCTGCA G 31 68 base pairs nucleic acid singlelinear cDNA NO NO 14 ACTGTCTAGA TCATAGGTCT TCTTCTGATA TTAGTTTTTGTTCCTTGCAC GAGGAGAAGG 60 TTTTCCAG 68 26 base pairs nucleic acid singlelinear cDNA NO NO 15 ATCGAGATCT GCCCTCCCCC TGGAGA 26 30 base pairsnucleic acid single linear cDNA NO NO 16 ACTGAATTCA GGCCACGGCTGCATTCACAG 30 6 amino acids amino acid linear protein internal 17 GlyLys Arg Pro Ser Ala 1 5 6 amino acids amino acid linear protein internal18 Gly Lys Trp Pro Ser Ala 1 5 6 amino acids amino acid linear proteininternal 19 Trp Trp His Glu Trp Ala 1 5 18 base pairs nucleic acidsingle linear cDNA NO NO 20 GGCAAGCGGC CGTCAGCC 18 18 base pairs nucleicacid single linear cDNA NO NO 21 GGCAAGTGGC CCTCAGCC 18 18 base pairsnucleic acid single linear cDNA NO NO 22 AGCCCACTCA TGCCACCA 18 15 aminoacids amino acid linear protein C-terminal 23 Tyr Pro Cys Lys Asn PhePhe Trp Lys Thr Phe Ser Ser Cys Lys 1 5 10 15 14 amino acids amino acidlinear protein C-terminal 24 Pro Cys Lys Asn Phe Phe Tyr Lys Thr Phe SerSer Cys Lys 1 5 10 701 base pairs nucleic acid unknown unknown cDNA NONO 25 CC AAAACATTGA TTTCAGGGCT GCCAGGAAGG AAGAGCAGCA GCAGGGTGGG 52AGAGAAGCTC CAGTCAGCCC ACAAGATGCC ATTGTCCCCC GGCCTCCTGC TGCTGCTGCT 112CTCCGGGGCC ACGGCCACCG CTGCCCTGCC CCTGGAGGGT GGCCCCACCG GCCGAGACAG 172CGAGCATATG CAGGAAGCGG CAGGAATAAG GAAAAGCAGC CTCCTGACTT TCCTCGCTTG 232GTGGTTTGAG TGGACCTCCC AGGCCAGTGC CGGGCCCCTC ATAGGAGAGG AAGCCCGGGA 292GGTGGCCAGG CGGCAGGAAG GCGCACCCCC CCAGCAATCC GCGCGCCGGG ACAGAATGCC 352CTGCAGGAAC TTCTTCTGGA AGACCTTCTC CTCCTGCAAA TAAAACCTCA CCCATGAATG 412CTCACGCAAG TGTAATGACA GACCTGAATA AAATGTATTA AGCAGCAGTG ATCTTTCCTC 472TCCTCCTTCC CAAGTCATTG AAAAGTGTTT GTTATTTAAA TTCCAATAAT GCCCAATACT 532GACGTGTCTT GAGTAATTTG GAACCCAAAA GTGAAGATCT TTGATAAAGA TTTTTTTTGT 592GGTTCGACTG GACTGTGCTG AGTGCGGGCA CTGGGCTTTT CTTCTGATGT TCATTATGGT 652GCTGGGAAGC TCTGTCTTTG ATTTAAAATA AAATAGCTAA AGGCTACAC 701 105 aminoacids amino acid single linear peptide NO NO internal 26 Met Pro Leu SerPro Gly Leu Leu Leu Leu Leu Leu Ser Gly Ala Thr 1 5 10 15 Ala Thr AlaAla Leu Pro Leu Glu Gly Gly Pro Thr Gly Arg Asp Ser 20 25 30 Glu His MetGln Glu Ala Ala Gly Ile Arg Lys Ser Ser Leu Leu Thr 35 40 45 Phe Leu AlaTrp Trp Phe Glu Trp Thr Ser Gln Ala Ser Ala Gly Pro 50 55 60 Leu Ile GlyGlu Glu Ala Arg Glu Val Ala Arg Arg Gln Glu Gly Ala 65 70 75 80 Pro ProGln Gln Ser Ala Arg Arg Asp Arg Met Pro Cys Arg Asn Phe 85 90 95 Phe TrpLys Thr Phe Ser Ser Cys Lys 100 105

What is claimed is:
 1. A substantially isolated and purified cortistatincomprising a sequence selected from the group consisting of SEQ ID NOs2, 5, 6, 7, 8, 9, 10, 11, 12, 23, 24, and
 26. 2. A substantiallyisolated and purified human cortistatin having at least about 95% aminoacid residue similarity with a cortistatin having a sequence of SEQ IDNO: 26, and inducing cortical slow-wave sleep isoform two.
 3. Thecortistatin of claim 2 wherein said human cortistatin amino acidsequence and the sequence of SEQ ID NO 26 are at least about 98%identical.
 4. A substantially isolated and purified cortistatincomprising a sequence selected from the group consisting of SEQ ID NOs26, positions 44 to 74 of SEQ ID NO 26, positions 77 to 105 of SEQ ID NO26, and positions 89-105 of SEQ ID NO 26, and inducing corticalslow-wave sleep isoform two.